some m m i m

c o n c m s m a pyrimidine biosynthesis

By
Robert L. Herrmann

A THESIS

Submitted to the School of Graduate Studies
of Miohigan State University of
agriculture and applied Science
in partial fulfillment of the
requirements for the degree of
DOCTOR OF PHILOSOPHY
Department of Chemistry

1956

ProQuest Number: 10008510

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/-

" ±r 't

')t y b

ACKI'iOWLKDGMEN'T

fhe author wishes to express his deep
appreciation to Dr* James &«. Fairley for his
interest* patience* and thoughtstimulating
guidance, which greatly facilitated the com­
pletion of this problem# He is also greatly
indebted to Dr# Richard C# Byerrum for his
interest and counsel#
The author also wishes to express his
appreciation to the various other members
of the Chemistry Department for assistance
and helpful suggestions#
Finally# the writer wishes to thank
the Atomic Energy Commission and Michigan
State University for providing funds in
support of this work#

ii

VITA
The author was b o m July 17, 1928 la lew York
City, and received hie secondary education at Bayside
High School, Bayside, M m York*

Be enrolled at Purdue

University for the spring semester, 1946, and entered
the United State® Havy la August of the same year*
After two years as a naval electronics technician
the author resumed his studies at Purdue University
and was graduated in June of 1951 with a Bachelor
of Science degree*

He was then recalled to naval

service for fifteen months*

In September of 1952

he enrolled in the Graduate School of Michigan State
university.

In the course of his graduate training

the author served two years as a Graduate Teaching
Assistant in Chemistry and two years as a Special
Graduate Research Assistant under an Atomic Energy
Commission Grant*

ill

SOME STUDIES COMOERHIBO FYHIMIDIHE BIOSYNTHESIS

By
Robert L* Herrmann

m

ABSTRACT

Submitted to the School of Graduate Studies
of Michigan State University of
Agriculture and Applied Science
In partial fulfillment of the
requirements for the degree of
DOCTOR OF PHILOSOPHY
Department of Chemistry

1956
Approved
*r-

iv

ABSTRACT
The following investigations were carried out to study
several aspects of pyrimidine biosynthesist

1) A study of

methionine methyl group utilization for thymine biosynthesis
in the ratj

2)

A study of the possibility of alpha-amino-

butyric acid utilization for pyrimidine biosynthesis in
HeuroBpora; 5 ) a similar study of aminobutyric acid
utilization in the rat*
Methionine-methy1-G14 was administered to rats and
the purines and pyrimidines of deoxyribonucleic acid were
Isolated and their specific activities determined*
Adenine, guanine and thymine were found to be appreciably
labeled, presumably by way of the "on©-carbon pool**

the

radioactivity of thymine appeared to be in the methyl
carbon, since cytosine was not labeled appreciably and the
iodoform - representing the methyl carbon - obtained on
degradation of thymine was highly radioactive*

The data

suggests that formic acid is not an intermediate in con­
version of the methionine methyl group to thymine# but
rather that a hydroxym ethyl derivative is involved*

The

results indicate that the methyl group of methionine is
not a significant precursor of the ureide carbons of the
purines, but its important position in biosynthesis of the
methyl carbon of thymine is definitely established*
A pyrimidine-requiring mutant of leurospora crassa.
strain 1298, which had previously been shown to utilize
v

alpha-aminobutyri o a eld for growth, was grown on the 3 -C*-4
labeled amino acid in an effort to determine the reason
for its pyrimidine-replacing ability*

The isolated ribo­

nucleotides were hydrolysed to the free purine and pyrimi­
dine bases* and the pyrimidines were found to have five
times the specific activity of the purines*

A similar dis­

tribution of activity was found in the acid-soluble nucleo­
tide fraction*

A number of amino acids isolated from the

soluble protein fraction were found to have specific activi­
ties even lower than the purines, with the exception of iso­
leucine, which was labeled to about the same extent as the
pyrimidines*

The fact that labeled aminobutyric acid - a

known intermediate in isoleucine biosynthesis - gave rise
to a similar degree of labeling in isoleuoine and the
pyrimidines establishes the importance of aminobutyric acid
as a pyrimidine precursor In Meurosoora* A ten-fold dilu­
tion of the labeled precursor was found to occur on conver­
sion to the pyrimidines, but a pool of diluted aminobutyric
acid was found to exist in the mold, suggesting that once
growth begins the organism is able to synthesis© amino­
butyric acid,

This phenomenon and the nature of the muta­

tion involved are discussed*

A possible pathway of utiliza­

tion of aminobutyric acid for pyrimidine biosynthesis in­
volving homoserine and beta-aspartyl phosphate is also
suggested*

vi

A study of the possible utilisation of aminobutyric
acid for pyrimidine biosynthesis in the rat was also begun,
the cytosine from isolated deoxyribonucleic acid was found
to be four times as radioactive as adenine from the same
source, suggesting that aminobutyric acid may also be a
pyrimidine precursor in the rat#

the low labeling of

thymine indicates that a different pathway for its bio­
synthesis may exist#

However, the generally low level of

radioactivity of the various Isolated compounds makes the
interpretation of the data of questionable value#

vii

TABLE OF GOMTENTS
Page

IRTRGBUGTIQM * ............... , ........... . . • . X
EXPERIMENTAL ARB RESULTS........ • , .......... • . 10
The Utilization of Methionine for ‘Thymine Bio­
synthesis • » « «
.............
10
Materials * • • • * • • • • • * • * • * * * * * * 10
Isolation of Deoxyribonucleic Acid • • * • • • • 1 1
Isolation of Purine and Pyrimidine Bases • • • • 15
Cytosine Purification • • • • * • • * • • • • • 1 6
Isotope Measurement* • • * • • • • « • • • • . « 16

Thymine Degradation* • « . » . . . . . • * * * * 1 7
Criteria of Purity * • • • • • • • • • • • • • • 2 0
Results* • • * * • • • • « • • • • • • • « • • •

SI

The Utilisation of Aminobutyric Acid for Pyrimidine
Biosynthesis in Reurospora* • • « • • • • • • • • • S4
Materials* • • • • • • • • * • • • • • • • • • • 2 4
Organisms* • • • • • * • • • • • • • • • • • • • 2 4
Growth Procedure*•
25
Overall Distribution of Isotope* • • * • • • • • 2 6
Study of Ribonucleotides • • • • • • • • • • • • 2 8
Isolation of Ribonucleotides* • • • • • • • • 2 8
Hydrolysis of Ribonucleotides • • « • • • • • 29
Isolation of the Purine and Pyrimidine Bases• 30
Purification of Purine and Pyrimidine Bases • 51
Recrystallisation • • • • • • * • • • • • 3 1
One-dimensional Paper Chromatography • • • 33
Specific Activity Determination . . . . . . . 55
Localisation of isotope*. • • • • • • • • • • 3 5
Study of the trichloroacetic Acid Extract, . . • 41
Isolation of Acidesoluble Nucleotides from
the TGA extract • • • ■ • • • • • • • • • • • 4 1
Two-dimensional Chromatography. . . . . . . . 42
Ion-exchange Chromatography* . . • • • • • • 4 4
Separation of a Uridine Rucleotide* * • • « • 45
Characterisation of Uridine-5*-phosphate• • « 48
Separation of an Adenine Nucleotide . . . . . 51
Characterization of Adenosine-5 -phosphate. • 53
Radioautography of Paper Chromatograms. . * . 54
Isolation of Amino acids from the TCA extract 55
Study of the Amino acids of the Soluble Proteins 56
Isolation and Hydrolysis of the Soluble
Proteins. * • * • • • • ........... . • • • 5 6
Separation and Purification of Protein
Amino Acids • • * ♦ • • • • • • • • * • • • • 5 7

Page
Study of the Final Nutrient Media • « • •
Study of the Saponifiable Lipid Fraction*
Study of ffeurespora Deoxyribonucleic Acid
The Effects or Related Substances in
Growth of the Reurosoora Mutant • « • •
The Utilization of Aminobutyric Acid for
Pyrimidine Biosynthesis in the Rat * * * • •
Materials* * • • « • • • • « * •
***••
Isolation of Deoxyribonucleic Acid* * • • •
Isolation of Purine and Pyrimidine Bases* «
Purification of the Purine and Pyrimidine
B a s e s * ........* * * * * ............ *
Concentration and Radioactivity Measure­
ments • « • • « * • • • • * • • • • • • •
Results * * * * * * * * * * • • * • « • * •
DISCUSSION* * • • . « • * . * • * • • * ........
The Utilization of Methionine for Thymine Bio­
synthesis* . . . . . . . i t . ...........
The Utilization of Aminobutyric Acid for
Pyrimidine Biosynthesis la Heurospora, * * .
The Utilization of Aminobutyric 'Acid'1for
Pyrimidine Biosynthesis in the Rat . * * * «

. 69
« 70
* 71
• 73
79
79
79
79
79
80
80
81
81
86

100

LIST OF TABLES
TABLE
I
IX
III

IV
V

VI

VII
VIII

PAGE
Incorporation of Formic Aoid-C14 and MethionineMethyl-C*4 Into Bat M A components* « * . • « 22
The Composition of the Basal Nutrient Medium* •

25

Distribution of Carbon-14 in ITeuros oora after
Growth in the presence of Aminobutyric Aeid3-C14* * * * * * • • * » « • • *
* * ****

27

Distribution of Carbon-14 in Uracil * * • • • •

40

Carbon-14 Content of Compounds of Neurosoora
after 4 days Growth in the presence of Aminobutyric Aeid-3-Cl4. , • • * • • • • • « • • •

67

Carbon-14 Content of Compounds of Meuroapora
1298 after 6 days Growth in the presence of
Aminobutyric Aeid-3-C^4 • • • • • • • • • • •

68

The Effect of Various Compounds on Growth of
Neurospora 1298* « * • *
. . . . . . . . . .

77

incorporation of Aminobutyric Aeid-3-C14 into
Bat DBA Components « • • • • * • * * • * • • •

80

XMmomQtWM

INTRODUCTION
The biosynthesis of the pyrimidine bases of the
nuclei© acids - the major constituents of the genetic
material of the cell and the active principle of virus
particle® - has become an increasingly important subject
for research studies*

A clear picture of the method of

pyrimidine formation by the ©ell has become a necessary
goal for any general under®tending of nucleic acid bio­
synthesis*
Early work on the origin of the carbon atom® of the
pyrimidine ring wa® carried out by Heinrich and Wilson (1)
who demonstrated the origin of the carbon atom at position
2 from carbon dioxide in the rat*

Similar results were

also obtained by Reichard and Lagerkvist (2)•
Work by Mitchell and Iioulahan (3) pointed to a role
for the 4-carbon acids of the citric acid cycle, since
oxalacetic acid and aminofumarlc acid were found effect­
ive in stimulating the growth of pyrimidineless 1 euros pora mutants*

The findings of Loring and Pierce (4),

showing that nucleosides were utilised more effectively
than the free pyrimidine base® by these mutants, suggested
that an acyclic intermediate may be combined with ribose
at some step prior to ring closure*

Orotic acid and

uracil were visualized as being utilized by way of ring
rupture and subsequent ribosidation, and Mitchell et al. (5),

2

after genetic investigations, arrived at the conclusion
that orotic acid was not a nowaal intermediate, but arose
In & side reaction during pyrimidine biosynthesis*
however* in other orgaaisms the importance of orotic
acid seems well established*

Reichard (6 ) found that

nl&«Xabeled orotic acid was extensively Incorporated into
the pyrimidines of polynucleotides of several organs of
the rat, whereas purines were not labeled*

hater work

by Weed and Wilson (7) corroborated these findings, since
orotic acid labeled in the @ position with C*4 was found
to be utilised similarly in the rat*

It is interesting

to note that the emtio acid pathway apparently exists
also in yeast, as shown by Edmonds, Delluva, and Wilson (8 )*
Experiments were also carried out with Lactobacillus bulgaricua 09 by Wright et al# (9, 10) which demonstrated
the requirement of this organism for orotic acid5 the re­
quirement could not be replaced by any other pyrimidine.
In addition, further understanding of the pyrimidine bio­
synthetic pathway in this organism was afforded by the
finding that DL-ureidosuccinie acid labeled In the ureide
carbon was as effective a precursor for nucleic acid
pyrimidines as was orotic acid*

This relationship was

also demonstrated to exist in the rat by Weed and Wilson (11)
who found that DL-ureidosuccinic acid was incorporated into
polynucleotide pyrimidines.
A role for aspartic acid as a precursor of the c&rbaa
chain of pyrimidines was suggested by the work of

3

Lagerkvist jjb al* (12), who tested aspartic acid-S-cl3*4«C14
in rat liver slice*# fhe labels were incorporated into the
polynucleotide pyrimidine*, though the methylene carbon wae
utilised to a greater degree than the carboxyl carbon which
suggested cleavage of the carbon chain in the course of
utilization*

The authors felt, however, that the molecule

wae used as a whole, since the pyrimidines were not de­
graded end there was thus some question as to the validity
of the Q » to C** ratio*

The importance of aspartic aeld

was also suggested by the experiments of ftoods, Havel and
Shlve (13) with hactobacilius arablnosus 17-5, an aspartic
acid-requiring mutant which was shown to be able to utilize
pyrimidines, as well as threonine and lysine, as a means of
sparing the aspartic acid requirement*

This was taken as

proof that aspartic acid was Involved in pyrlmidino bio­
synthesis#
The conversion of I*~aspartie acid to L-ureidosueclnic
acid in rat liver mitochondria has recently been demon­
strated by Reichard (14)«

Aspartic acid, carbon dioxide

and ammonia are converted to ureldosuc cinic acid in the
presence of carbamylglutamic acid, adenosine triphosphate,
and magnesium ion*

The reaction appears to require several

enzymes, and the Initial step may be formation of carbamyl
phosphate• The latter has been demonstrated as involved in
the conversion of aspartic acid to ureidosuccinic acid in
S* fecaells extracts by Jones, Specter, and Lipmann (15)*

4

She entire sequence of reactions from aspartic acid
to pyrimidines has been demonstrated by hiebenaan and
Komberg (16, 17, IQ) for %ymobacteriu» orotlcum. An
enzyme called ureidosuccinase converts L-ureidosucoinic
acid (XI) to 1 -aspartie acid (X) with the liberation of
carbon dioxide and ammonia*

The next step is reversible

and involves cycllsation and dehydration to L-dihydroorotie acid (XXX) with the involvement of an enzyme called
dihydroorotase• The dihydropyrimidine is then oxidized
to orotic acid (IV) by the action of dlhydroorotie de­
hydrogenase*

The conversion of aspartic acid to urel&o-

succinic acid appears to require a separate enzyme system,
perhaps involving an enzyme such as that found in rat
liver extracts by Lowenstein and Cohen (19).

This enzyme

may convert carbon dioxide, ammonia, and adenosine triphos­
phate to a reactive oarbox&mide-group donor such as oarb&myl phosphate, which then reacts with aspartic acid to
form ureidoiuceihi© acid.
COgH

lion

I
H-C-H

(
H

|
h

i

-M * C-COgH

M

0»C

I

<* C-COgH

I
H

L-dihydroorotic
acid
III

C*Q

I I

I I

H

II

H-H -

0*»G H - C - H

|

I**.asparti©
L-ureldoacid
succinic acid
I

H - H - C* 0

I I

0 »G H - G - H

I
H^-C-COgH

GOoH

I I

C-H

M

H»*f *

I

(•

COgH

orotic acid
IV

6

The existence of the orotic acid pathway implies the
initial formation of the pyrimidine ring followed by rlbosidatloa, end this sequence of reactions wae recently shown
by ULeberstan, Komberg, end Simms (20)»

Yeast ensymes were

able to convert orotic sold to ©roiidine*S* -phosphate (V)
b y reaction with 6 *phosph©rlboayIpyrophosChafes, and then,

by d« earboxylat ion, to convert eret idine*6 *~phos isfeate to
uridime*b*<*phoephate (Vi)*
m

I

* e»o
I

M f * C«0

<3*1

<M5
C~H
I
II
h *i * c*a

G»C

I

II

11*1 » C*G0g!I

I*G---I
a*swoe
i

o

I*G*0M
I
11*0^0

I

II

I
II
H«CU9*?~0H
I
0H

%C*0*fM>S
I

01
orotidine-b*-phosphate

urid iae*5 *-phoa phate

f
m

contrast to this rather well*dsfinad biosynthetic

pathway in several bacteria, in yeasts, and la the rat,
pyrimidine biosynthesis in leurosoora is not clearly under*
stood.

The *orb of Mitchell and Heulahan (3) and of Loring

and fierce (4) combine to suggest a role for some acyclic
rlboee derivative.

The occurrence of a second possible

pathway does not see® unlikely since the same situation

6

exists la purine biosynthesis, where much work by Buchanan (ZL)
and by Greenberg (22 ) has established an acyclic ribose deri­
vative as an intermediate in purine biosynthesis in pigeon
liver.

However, Kornberg and coworkers (23) have recently

reported the reaction of purines with b-phosphyribosylpyrophosphate to form the nucleotide in yeast extracts.

She

two mechanisms, ribosldatlon before and after ring closure,
therefore do occur, and it seems reasonable to suspect that
a similar situation exists in pyrimidine biosynthesis.
Furthermore, a number of growth studies also suggest
that some organisms may possess a pyrimidine biosynthetic
route distinct from that involving orotic acid.

Woods and

coworkers (13) were unable to replace the aspartic acid re­
quirement of hactobaclllus arabinoaua 17-5 by either orotic
acid or ureidosuccinic acid.

Jh addition, Fairley (24)

has demonstrated that several pyrimidineless Neurospora
mutants may utilize threonine (VIII) or alpha-aminobutyric
acid (VIII) for growth.

Aspartic acid was ineffective, and

later work (25) demonstrated that ureidosuccinic acid was
likewise not utilized.
hater, evidence was obtained for the involvement of
the “l-carbon-pool" in the biosynthesis of the 5-methyl
carbon atom of thymine by Totter et gl. (26), who found
activity In this group using formic aeid-G*4 as precursor,
and by Elwyn and Sprinson (27), who demonstrated the utili­
zation of serine-3-Cl4 and glycine-2-C14 for this methyl
carbon.

The Involvement of metaionine (IX) in the

7

"1 -carbon pool11 «&e indicated by the work of Borg (£8 ),
which demonstrated the c ©aversion of formic acid to the
methyl group of methionine la pigeon liver extracts, and
suggested an S-hydroxymethy 1 derivative as an intermediate*
In addition, methionine has been shown to be a very Import­
ant donor of intact methyl groups in transmethylation reac­
tions in animals (29)*
$8®

C H g —8 — Chg

H-O-OH

H-C-H

fUC~H

r

r

i

I
CGvrE
Xi-threonlne

711

H»C-KH0
I
2
COjgH

B-C-lHp
I
COgE

k-alpfea-amitio-nbutyric acid

L-methlonine

7111

ix

It therefore appeared likely that methionine might be

a significant precursor of the methyl group of thymine (X),
by way of the ®l-earb©» pool®, and the possibility also
existed that a transmethylation to the $ carbon of the
pyrimidine ring might be a elgnifleant source of the thymine
methyl group.

In the present work tee possible utilisation

©f Gi4&-methyl-labeled methionine for the biosynthesis of
the thymine methyl carbon of deoxyribonucleic acid in the
rat has therefore been investigated*

the labeling of the

purine bases, adenine (XI) and guexxine (XII), of deoxyribo­
nucleic acid was also studied as a possible means of com­
parison of the Importance of the two possibilities -

a

transmethylation, or oxidation to a 1 -carbon intermediate and fomie acid-C^ was run In parallel experiments to have
a means of comparison with other published work.
H-N - C—0

I I
0*C

G-C%

I I
H-K - C-H

thymine
X

U - G-NH©
I I
H-C

H-H * C-0

I I
C-Jf.

I I

Hgl-C

\-H

If * C -W

adenine

I

C-H.

I \j~H

M - G-H

guanine

XI

The previously-mentioned growth studies in which
alpha-aminobutyric aold was found to support the growth
of a pyrimldineless mutant, N. crassa strain 1898, suggest­
ed that this amino acid might be involved in pyrimidine bio­
synthesis In &eurospora. The fact that the generally accept*
ed pathway - involving conversion of aspartic acid to the
pyrimidine nucleotides by way of ureidosuccinic acid, dihydroorotic acid, and orotic acid - does not appear to exist
in this organism seems to support this possibility.

The

present work therefore also Includes a study of the label­
ing of the pyrimidines - uracil (XIII) and cybosin© (XIV) and related compounds after growth of I. crassa 1298 on
5 - C ^ alpha-aminobutyrie acid,

A similar study has also

been started with the rat.
Growth studies with Jieurospora were also carried out
to test a number of possible relationships suggested by
this work and by recent reports of Fink, ert al. (30)
and of Grisolia and Wallaeh (31) that a biosynthetic

9

pathway Involving conversion of beta-alanin© (XV) to beta*
ureidopropionio acid (XVI) and subsequent eyclisation may
be involved In pyrimidine biosynthesis*
H -8 - 0-0
I

I

0-0
C-H
I
II
H-JS - C-H
uracil

H-H - C-h%
r
I
0-C
C-H
I
II
H - C-H
cytosine
XIV

XIII
0 0 #

CHg

0—0
I

%h-ch 2
beta-alanine
XV

OH©
I

H-H - GHg
beta-ureldo
propionic acid
XVI

EXPERIMENTAL AND BESULfS

EXPERIMENTAL M D RESULTS
Utilisation of Mthlontno

i 2E gtefeft Mosynthesla
Materials
fw© male albino rats weighing about 160 g« were in­
jected intraperitoneally with 1 ml* of a water solution
containing 0*1 mo* of methtonine-roetiyl-cl4,

a second

pair of rats were injected with 0*1 mo* of formic acid-C*4**
This amount of activity was supplied by 20 mg* of radio­
active methionine and by 2*61 mg* of formic acid-Ci4*
The synthesis of methloaine-methyl-C14 was carried
out according to the procedure of du Vigneaud, Dyer, and
Harmon (62) • This consisted of the reduction of 1 milli­
mole of homocystine to homocysteine by reaction with sodium
in liquid ammonia*

The mixture wae contained in a glass

tube through which nitrogen gas wae slowly bubbled to
afford stirring and to maintain an inert atmosphere.

A

temperature of approximately -?0®C was maintained by use
of a dry lee • acetone bath*

One millimole of C^4-methyl

iodide (1 mc./mM. activity) was added to the reaction mix­
ture after warming to room temperature*

The mixture was

concentrated to a small volume, and the resulting methloninemethyl-C14 crystallised out*

The crystals were washed with

^Obtained from the Isotopes Specialties Company,
Glendale, California

11

Hold ethanol* then with ether, and dried in vacuo.

The

yield after recrystallization from 80 per cent ethanol was
90 per seat of theoretical#

The compound melted at

279-28Q°C*
Isolation of Deoxyribonucleic Acid
The rats were killed toy etherization 24 hours after
injection.

The total viscera* including hear fc, lungs*

kidneys* liver* spleen* stomach* intestines and testes*
was then removed* cleaned* frozen on solid carbon dioxide*
and stored la the deep freeze for 12 hours*

The frozen

tissue was diced and then homogenized in a Waring Blender
with 209 ml* of cold absolute ethanol for five minutes*
The hemogeaate was transferred to 280 ml* Pyrex e entrifuge bottles and centrifuged at -10°C for 20 minutes at
@000 r*p*m*t using a Model V International centrifuge «
The supernatant was discarded and the residue was extract­
ed three times with a helling 3:1 {v/vj ethanol-ether mix­
ture for five minutes*

The residue from these lipid ex­

tractions was weened twice with ethanol* three times with
ether* and was then air dried*
The dried* lipid-extracted viscera was then placed in
a mortar and mixed with ten per cent (w/v) sodium chloride
solution to make a paste*

Carborundum of 120 mesh size was

then added and grinding carried out for 15 minutes • The
mixture was transferred to a 250 ml* Pyrex centrifuge
bottle, using a sufficient amount of the sodium chloride
solution to make a final volume of 150 ml*

The mixture

12

*11# then heated on the steam bath fox* 20 minutes # and there­
after stirred slowly for 24 hours at room temperature*
After eentrifuga tion, the remaining solid was re-extracted
with a 100 ml# portion of fresh sodium chloride solution
for 12 hours# and the extracts combined• The residue from
the re-extraction was washed with 20 ml* of the sodium
chloride solution and the washing added to the combined
extracts la a 1 liter beaker*

Two and one-half volumes

of ethanol were then added to precipitate the crude sodium
nucleates# which were centrifuged down in 260 ml. centri­
fuge bottle#*

After washing with ethanol and ether# the

material was air dried and weighed * A typical yield was
1 g, of mixed nucleates.
Deoxyribonucleic acid was Isolated from the mixed
sodium nucleates by the method of Hammers ten (33) *
Sufficient 0.1 I sodium hydroxide was added to the dried
nucleates in a 200 ml* flask to make a 1 per cent solu­
tion*

This was then heated in a boiling water bath for

two hours and acidified to pH 2 with 2 I hydrochloric acid.
One-tenth volume of 0*1 M lanthanum nitrate was then added
to form the insoluble lanthanum salt of deoxyribonucleic
add# and the mixture was centrifuged in 2 50 ml* centrifuge
bottles•

The precipitate was washed twice with small

amounts of 0.Q1 M lanthanum nitrate# transferring the solid
to a 15 ml. centrifuge tube in the process. The lanthanum
deoxynueleafce precipitate was finally treated for separation
from contaminating protein by the following procedure*

One

13

ml# of 1 M potassium carbonate and four and one-half ml*
of water was added to the solid, and the mixtore heated in
a hot water bath for 5 minute® • After centrifugation, the
eolation was placed in a 100 ml* beaker and the precipitate
re-extracted twice with 0.6 ml* of water*

The combined

supernatant® were acidified to pH 5.7 or below with glacial
acetic acid, and then boiled for five minute® to remove
carbon dioxide*

The deoxyribonucleic acid was precipitated

by the addition of four volume® of ethanol*

The mixture

wae placed in the refrigerator for 18 hour®, after which
it wae centrifuged, and the greyish-white precipitate
washed with ethanol, ether, and then air dried and weighed*
The average yield from a 160 g* rat wa© about 100 mg, of
deoxyrlbonuole i© a©id *
Isolation of Purine and Pyrimidine Bases
The deoxyribonucleic acid was placed in a 10 ml*
volumetric flask and hydrolysed by the method of Marshak
Vogel (54),

Two ml* ©f 7 N perchloric acid was added

and the mixture was heated on the steam bath behind an
explosion shield for one hour with occasional shaking*
The content© of the flask were then transferred to a 15 ml,
centrifuge tube with 1 ml* of water, and the charred resi­
due washed with one 0*5 ml, portion of water*

The combined

supernatants were placed in a second 15 ml* centrifuge tube
and baeified to a pH of 11 by tho addition of 5 S potassium
hydroxide*

The precipitate of potassium perchlorate was

14

removed by centrifugation, and the Bupematant containing
the free purine and pyrimidine bases, usually an ambercolored liquid due t© the presence of suspended colloidal
carbon particles, was placed on a 2*5 by 27 cm* Mlseo*
©oilman packed with Dowex 1 anion exchange resin of 50 to
100 mesh size and twelve per cent croselinking*

The resin

had previously been converted to the chloride form by treat­
ment with 1 I hydrochloric acid and subsequent washing to
remove excess acid*

After the solution of bases had fil­

tered Into the resin, a chromatographic procedure similar
to that suggested by Cohn (55) was carried out*

The pyri­

midine bases were eluted in separate peaks by elution at
the rate of two and one-half ml* per minute with 0*015 M
ammonium format© buffer* of successively lower pH*

The

first buffer to be passed through the column was of pH
10*1, and served to ©lute eyfcosin© in a 75 ml* peak after
about 100 ml* of buffer had passed through the column*

The

samples were collected in 50 ml* beakers located on the
turntable of a Misco automatic fraction collector, and the
elution of the samples was followed by reading a portion
of the contents of each beaker in a Beckman Model DU
Spectrophotometer****

The ratio of optical density

(absorbancy) at 260 mp. to that at 280 ®p was found most
reliable as a means of following the elution of the bases

*Mioroohemical Specialties Co*, Berkeley, California
**Hstional Technical Laboratories, S* Pasadena, Calitteda

15

from the column.

After elution ©f cytosine, the column

was next treated with about 150 ml. of buffer of pH 9 .1 ,
which was then followed by addition of buffer of pH 8*95
for the removal of thymine*

After the passage of about

160 ml# of the buffer, the pyrimidine appeared as a broad,
symmetrical peak in 200 to 260 ml# of effluent.

Finally,

adenine and guanine were recovered by addition of 200 ml.
of 1 If hydrochloric acid*

The acid effluent was taken to

dryness several times In vacuo to remove excess hydro­
chloric acid, and the residue, taken up in a few ml* of
0*1 M hydrochloric acid, was allowed to filter into the
resin bed of a 1 by 12 cm* column.

The resin used was a

Oowex 50 cation exchanger of twelve per cent cross linking
and 100 to 200 mesh particle size which had been placed
in the hydrogen form by successive acid and water washes
ae described for the preparation of the Dowex 1 column.
The purine bases were ©luted with 5 N hydrochloric acid,
guanine coming off in a 200 ml. volume after 50 ml. of
eluting agent had passed through the column, and adenine
following - after efflux of an additional 40 ml. of acid in a 200 ml. volume• Th© elution of the bases was followed
by observation of the ratio of optical densities at 249
and 260 mu for each fraction.

16

Cytosine Purification
The ratio of optical densities at two wavelengths of
the cytosine fraction, when compared to the ratio for known
eytoslne, indicated considerable contamination, and the
eluate was therefore evaporated to a small volume, soldi*
fled with 2 drops of concentrated hydrochloric acid, and
placed on a 1*3 by 23 cm* column packed with Dowex 50
resin in hydrogen form*

After allowing the sample to

filter in, the column was treated with 2 H hydrochloric
acid, again following elution of the cytosine by observa­
tion of the optical densities at 260 and 280 mp*

A value

of 1*55 for the ratio of optical density at 280 mji to
that at 260 up indicated that the cytosine was satisfac­
torily pure*
Isotope Measurement
One ml, aliquots of the thymine fractions obtained
from the original Dowex 1 separation and similar aliquots
of the cytosine fractions obtained from the Dowex 58
purification were plated on platinum dishes*

Half-

milliliter aliquots of the purine fractions were simi­
larly plated•

M

all cases the solvents were removed by

slow evaporation over an infrared lamp, and the radio­
activity of the samples determined at infinite thinness
in a Nuclear internal flow Geiger counter.

Since the con­

centration of each aliquot was known from optical density
measurements and molar extinction coefficients, the specific

17

activity* expressed as counts pap minute pap micromole,
could Pa calculated*

The molar extinction coefficients

for thymine In 0*015 M ammonium formate buffer and fop
adenine and guanine in 4 M hydrochloric acid and 3 N
hydrochloric acid respectively were determined as outlined
in the appendix*

The molar extinction coefficient of

cytosine in acid solution was obtained from the litera­
ture (36),
Thymine Degradation
2h an attempt to establish that the activity of the
thymine molecule actually resided in the methyl carbon,
the degradation of the molecule was undertaken*
The method of Baudisch and Davidson (37), was first
tried• This involved conversion of thymine to 5-bromo-4hydroxyhydrothymine with bromine water, followed by
hydrolysis in the presence of silver oxide to thymine
glycol and further hydrolysis with sodium bicarbonate to
urea plus acetol« The acetol, representing eaPbons 4 and
6 and the methyl carbon, was distilled out of the reac­
tion mixture and converted by iodine in sodium hydroxide
to glycolic acid and iodoform.

The latter represented the

original methyl carbon of thymine*

In practice the thymine

glycol was not isolated, the hydrolysis being carried out
directly to the acetol stage*

Some difficulty was exper­

ienced in obtaining favorable yields on the millimolar
scale by this method, and a more direct procedure,

18

suggested by Dr* John C* Speck of the Chemistry Department,
wafl found more useful,

This conaisted of direct reaction

of thymine with iodine in sodium bicarbonate solution,
and gave yields of eighty to eighty-five per cent of
theoretical#
Isolation of thymine from the column effluent was
found necessary as a preliminary to the degradation to
Iodoform*

Formate ion was found somewhat inhibitory,

probably by competition for hypoiodite ion, and ammonium
ion was strongly inhibitory at the concentration® of the
buffer, though studies of the latter effect indicated no
general inhibition of the iodoform reaction#

Experiments

to determine conditions under which sodium formate buffers
could be substituted for ammonium formate proved fruitless*
The eluate was also shaken with Dowex 30 resin in hydrogen
form in an attempt to remove ammonium ion, followed by a
similar treatment with Dowex 1 resin in iodide form to re­
move formate ion, but thymine still would not give iodo­
form*

It was finally decided to use a column of Dowex 50

resin, in the event that shaking of the resin with the
eluate did not bring about complete removal of interfering
lone, which were therefore present in sufficient quantity
to prevent reaction*

The procedure was successful in re­

moving ammonium ion, as evidenced by a negative test with
Kessler’s reagent, and evaporation of this column eluate
to dryness served to remove formate ion as volatile formic

no

ooto.

fli# U w m l m m m m m a t m m m * to « w

m U volwsaotFie

fXftftfc titfe ft mi* of bftlf«ootototod oodlim fttoorftottAto ioiu»
tton and too to ta l o f ft#91 ag* who

w ith to te * to io

Msoioit of toXatoXod topsino to ftio© o toton of 8*18 sg»

Out o l« « f a o o lo tlo n o f ito lo # to
f t lf t t t o i o f

lo d id o son*

g» o f wmM,i$m& io d to o In sift ml# ©f o o to f

© fto to to to i 00 $« ©f # § to i« te todto# ooo odood*

to o o to *

ftooo o»ft tofttaOotod oft 8?% * f o r *m# mmk# ood to# io fto *
fo m * Obion

m fo llo w p lo to lo to # woo fllto o o d

o f f m ft f r it o t o fXtoft ftx to o *
w tm ootovotod p u w i w

to o iM M ololtoto m& m»h&&

Sooldo « e ltttlo a to **osaoo« ©©©tom*

la o tla f lodltko* «cmI too oi^fttoX * to m dloftolvod tmm too
f ilt o F « lto
toFtd oototi
fo&a*

mA too o o ltttio a oooooootod m *
too to ta l ? lo ld woo 0*1 *ssg* o f iodo*

A ttoaotd to iwF&Xf too to t!o fo m by o u fe lliia tlflB tod

©rowed »ftu # fto # ftf8 l to t r i a l o*port«io«io ftto to f e # r » l do*
ooooooIUob to lad too* *o vlto oo ft fw otttor © w ftfio o tto o too
®mtp®wA m m % m k m m

to oiOorotowa m & w m * up to ft al*

to ft <i!o*«*#t©fpo*oO m t m m W l u float#
IXiftUfttft ragging from 100 to too isl* w o w ©I*tod on
ptottow diaboo# offttoOOtod to OFfftOftft, uto te o A to to ly
ooinbod to fto iatofoal floo

it was diftmvoFod

mm% tm ® fovm bad m ©ooootetoo oftUoo m to o oouBfeiog s'©to

of otuto 'MOftolttoo ttoftt too longor m m p l m had too© activity
tboo tiio mmllmw oaaploo*

ft too# too Xoogoot oatagfto ©Xatod

oftft * m lj 0#fX mg## too roodJLto oould o o t to fttto ib u to d to

20

self-absorption, so an attempt was made to obtain a quench­
ing curve of the amount of iodoform versus the activity of
the sample by plating known quantities of radioactive sodium
carbonate mixed with known amounts of unlabeled iodoform,
and determining the effect of the added Iodoform on the
counting rate*

This curve should supply a factor by which

the activities of corresponding amounts of labeled iodoform
could be multiplied to obtain the actual activity*

However,

when this procedure was applied to the radioactive iodoform
from the thymine of the methionine-fed rat, the specific
activity determined was double the largest value theoreti­
cally possible.

As an alternative, the remaining labeled

iodoform, amounting to about 0*5 mg*, was plated and an
attempt was made to count the sample with an end-window
Geiger counter, where no quenching is possible*

However,

the count obtained was not sufficiently above background
to be significant.

A similar result was found for iodo­

form from thymine isolated from a formate-fed rat*

Hhe

solution to this problem thus appeared to lie only in an
increase of the scale of the experiment®*
Criteria of Purity
The fractions obtained in the column separations of
the various bases were found to be satisfactorily pure as
judged by the ratio of optical densities at two wavelengths*
In the case of both cytosine and thymine the ratio of
optical density at 880 mji to that at 860 mp was used.

21

For cytosine the value Is 1*65, and for thymine the value
ie 0*66*

In the ease of the purines the ratio of optical

density at 249 mp. to that at 260 mp was employed, and the
values obtained were 1*3 for guanine and 0*81 for adenine*
In addition, a comparison of radioactivity and concen­
tration was also made#

Aliquots of the chromatographic

fractions which contained the labeled bases and also
aliquots of several fractions devoid of these compounds
were plated mi platinum dishes and counted*

The activi­

ties of these variotas fractions were plotted on the same
axes as a curve of concentration versus volume of eluant*
The coincidence of the radioactivity and concentration
curve® substantiated the purity of the Isolated purines
and pyrimidines in each ease.
Results
The radioactivity of the isolated bases of the
deoxyribonucleic acid of the rat after injection of formic
aoid-C^4 and methionine-methyl-C*4 is shown in Table I*
The specific activity was expressed as counts per minute
per micromole, the calculation of which is shown in the
appendix*
It can be seen that appreciable amounts of isotopio
carbon appeared in the adenine, guanine and thymine of
deoxyribonucleic acid after administration of either pre­
cursor*

The utilisation of methionine was poorer than

that of formic acid, as indicated by the dilution values

22

8 8

o o
HI HI

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a

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W

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H

CJ

H

H

H

g

iq

8

eu

H

%.

«

*0

HI
*
0

8

§
HI

H

O4k- »4k
o» q

ft

8
*

01

©
to

0*4
#
0

O 0
H
0H
40 e
"kJ

£
o*«0

m

hi

ih

<s

0
0* #4

s a
ft. ■

*

0

iO

o*
*
r*4
HI

4}
*4 +9
h d
Q r-f
©o
Pi ta
<ah

*-t

S

g

l

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n

»4

« ^

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H
o 6
Ps*

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43

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23

in the table*

However* It is seen that the methionine

precursor suffered a tenfold greater dilution on entering
the purines than did formic acid* but only a two-fold
greater dilution upon entering the thymine molecule.

If

the distribution of activity in the purines and thymine
after methionine administration may be considered similar
to that previously shown for formic acid* (5) the purines
should be labeled in carbons 2 and 8 and the major activi­
ty of thymine should reside In the methyl carbon*

The

low activity of the cytosine lends support to the latter
assumption.

'The results indicate that methionine does not

serve a® a significant precursor of the ureide carbons of
the purines* and therefore does not eontribute appreciably
to the pool of wl-carbon units® at the oxidation state of
formic acid.

In contrast* the relatively higher Incorpora­

tion of the methionine methyl group into thymine suggests
that conversion to a Bl-carbon unit® at the oxidation
state of formaldehyde may readily occur, and that this
derivative may then give rise to the methyl group of
thymine.

Transmethylation, though a possibility a® a

partial explanation of the data* cannot be a significant
mechanism for thymine methyl formation because of the
better utilisation of formic acid*

24

f&£ Itiiligatfen of Arninobufcyric Acid for
fyrimidine gfosynthe&fa tn Heuroseora
Material*
Radioactive alpha-aminobutyric acid, labeled with (A4
in the beta carbon atom, was kindly supplied by Dr* H* Tarver
of the "diversity of California#

Paper chromatography and

radioautography of the compound by procedures outlined in
a subsequent section (see pages 33 and 54) indicated that
the amino acid was essentially radiochemically pure, except
for a slight Indication of radioactive contamination which
migrated as glycine and whose concentration was estimated
at leas than a tenth of 1 per cent of the total activity.
The specific activity of the aiainobutyric acid was given
as 3*1 jcc/mg*, or 3*05 x 10® e*p*au^pM»

However, the value

actually obtained by quantitative ninhydrin (see page 64)
and radioactivity (see page 16) determinations of the
chromatographed sample was 4.41 x 10® e.p.m./pM.

In order

to obtain a pyrimidine labeling suitable for counting,
11 mg, of the radioactive compound was diluted with 89 mg.
of recrystallised unlabeled amlnobutyrie acid, and the
final activity of the precursor was therefore 4.84 x 104
c»p.m./pM«
Organisms
Henrosoora erases strain 1298, a pyrimidine-requiring
mutant which had been isolated by Beadte and Tatum (38)
and later described in detail by Lorlng and Fierce (4), was

25

m o d throughout the present study* except for several
experiments where the wild* non^mutated strain was employed#
The mold was maintained on culture slants consisting of
basal medium containing $ per cent agar and 1 mg# of uracil
per ml*
Growth Procedure
The mold was grown in 185 ml* Krlaomeyer flasks to
which was added 12*5 ml* of a basal nutrient medium having
the composition shows in Table IX*

The composition of

500 ml* of the stock trace element solution* of which only
50 pi# are used per 10 1* of media* is also included In the
table*
TABLE II
THE COMPOSITION OF THE BASAL HUTBIOTt MEDIUM

Calcium chloride
1 g#
Ammonium tartrate
SO g*
Ammonium nitrate
10 g#
Potassium dehydrogen
phosphate
10 g#
Magnesium sulfate
heptahydrate
5 g*
Sodium chloride
1 g#
Sucrose
101 g#
Biotin
26 jag.

Trace element solution §0 jxl.
Sodium tetraborate 8*8 g*
Ammonium molybd&te 6*4 g*
Ferric chloride
50 g#
&ine sulfate
heptahydrate
200 g#
Cuprie ihlfate
27 g*
Mangahbttr chloride 4.5 g*
Distilled water to 500 ml#
Distilled water
to
10 1*

Five mg* of the labeled alpha* amino butyric acid having a
specific activity of 48*000 c*p.m./pM and a total activity
of 2*55 x 10® counts was added to each flask, and the
flasks were then stoppered with cotton plugs and autoclaved

26

for 26 minutes*

Aft** cooling, the flasks were innoeulated

with 0*2 ml* of a spore suspension of the mold, made by
dispersing two loopfuls of the spores in 10 ml. of sterile
distilled water*

Incubation was usually carried out at 25°C

for 4 days, at which time sporulation was just commencing,
The contents of the flasks were then filtered on a fritted
glass funnel with suction* and the mycelial residue was
washed with 5 ml* of water and then soaked in 100 ml* of
acetone for IS minutes*

The acetone was removed and the

myeslia placed la a desiccator for 24 hours*

A brittle

disc was thus obtained which was suitable for grinding.
Overall distribution of Isotope
The amounts of activity contained in the mycelium, the
trichloroacetic acid and ethanol-ether extracts, the ribo­
nucleotides, and the residual growth medium were determined
in the following manner.
The total amount of activity contained In the mycelium
was determined by suspending 10 mg, of the total ground
mycelium in 6 ml* of water in a volumetric flask,

A 1 ml.

aliquot was then diluted to 100 ml., and 1 ml* aliquot® of
this diluted sample were then plated and counted as de­
scribed previously (see page 16).

One milliliter portion©

of the residual growth medium, obtained by filtration of
the mycelial pad© at the end of the growth period, and
similar portions of the trichloroacetic acid and ethanolether extracts, obtained as described In the following

27

section, were diluted to 10Q ml# with water#

One milli­

liter aliquots were again plated and counted#

The ribonu­

cleotide fraction was counted by diluting 100 pi* of the
ribonucleotides *• obtained by column purification as
described in the next section - to 5 ml*, and again plat­
ing and counting 1 ml* aliquots•
The results of the determination of the overall dis­
tribution of radiocarbon in the mutant and the wild strain
are compared in Table III*
TABLE III
DISTRIBUTION OF CARBON-14 IN N1UR0SP0BA
AFTER GROfTH IN THE PRESENCE
OF AMINOB'OTYBIC ACID~3-c£4
■-■■I-'-"..... ■■■■'■■ r -Trr. .r-..’AcTjVlly 'as''10*) 0 *0 .HU
Fraction or Components
Aminobutyric Aeid-3-<A4 supplied
Growth medium
Mycelium

Mutant Strain

Wild Strain

47.0

47*0

8*0

2*4

20*0

15*9

TCA-soluble*

8*0

4*0

Lipids

0*7

0*2

Ribonucleotides

2*8

1*7

11*8

10*0

Residue (by difference),
Sot accounted for
(loss as COg)

19.0

28*7

Soluble in 10 per cent trichloroacetic acid

28

Xt is interesting to not© the much higher activity contained
in the residual growth medium of the mutant, suggesting the
accumulation of one or more radioactive compounds« Perhaps
the two highly labeled amino acids located on radioautograms
of chromatograms of the growth medium are these compounds#
Study of Ribonucleotides
Isolation of Ribonucleotides» The mycelium obtained
from 10 flasks of the mold grown for 6 days on 3-C14aminobutyric acid was ground In a mortar with 120 mesh
carborundum powder for 15 minutes#

The resulting powder

was extracted 5 times with 10 ml# of cold, 10 per cent
(w/v) trichloroacetic acid transferring to a 12 ml, Pyrex
centrifuge tube in the process#

The solid residue was

washed once with 4 to 1 (v/v) ethanol and was then ex­
tracted 3 times with boiling 3 to 1 (v/v) ethanol-ether.
The extractions were carried out by suspending the centri­
fuge tube in a boiling water bath and stirring the contents
occasionally#

The process was carried out for a half-hour

the first time, and for 5 minutes the remaining times#

The

lipid-extracted residue was then washed twice with ether
and air dried#

Five milliliters of 1 N potassium hydroxide

was then added, and the mixture allowed to stand at room
temperature for 24 hours#

After centrifugation, the super­

natant was transferred to a second 12 ml# centrifuge tube
cooled on ice, and acidified to a pH of 3 with concentrated
perchloric acid*

The resulting precipitate of potassium

29

perchlorate

protoin wm allowed to coagulate for 10

miltube* ana tom m e rmomd by omtrifugaaoo. toe re*
*tilting eupematenfc of PibtoaeXeebtoee and twoponded protein
wa# filtered torough © layer ef ©elite on * fritted glee#
filter* end be#tried to « pa ©f u

with 1 I totoeelw

fjydwiM®, the ge&utlen of rllKMMieXeetidee tout obtained
« « o&Umwt to filter Into tifeyfi? *■« eel«n oonteining
®mm 1 ftxrten*e*«2»tog« mein of ftiMOO nrnm m%m end W
per ©«nt erroeUniting*

to# m t m m wee developed fivwt with

800 tit# of wetor# end t o m with too ml, of i a byds»«ta©Pi©
eoM*

toe option! deneity of toe letter eluate m » deter*

mined at tiO uu* end an epprewtotto eetinefclea eeeffieiont
of 10*090 together with <*n average aieleeul&r weight of M l
m e need to eetlMto toe «*B4Mttt*atien of mim&
imi 0 mg*

fhe oolutlan wee then ev*fMf*te& In m e n © to dry*

no** eeweoel tlnee to remove hydremlerle eo&d yielding *
gywletomfelte eeoidwe#
tc Il^aiiy^lliii*

* * « * « * ribonueieo-

tide# wore token op to eeeetel id** of 0*1 ubydro-ehlorfto
told end. ttneofewred to a gla**«oto&pe*e&» 10
stotrie fleefe* toe elate*# m e t o m blewst to dvynoos by
sseene of » *t*wm of eheoeeel-fUtered air* After easeful
addition of 0*8 ml* of eeneentpafced porohlerl# aeift* to#
t'lmk mm d m tod m toe otoott bato bobtnd an eeplosion
toield for 40 mlnufcea* toe ©entente of the fleek were toon
tmneferred to a It

ml,

eentrlfwge tube with the aid of

so

several small portions of water, and, after centrifugation
and transfer of the supernatant to a S %lf beaker, the pre­
cipitate was washed and the washings added to the beaker,
The solution was then diluted to about 5 ml., to provide
the perchloric aeid concentration of about 1 H, preparatory
to separation of the mixed bases*
leelatlon of the Purine and pyrimidine Bases * The
solution of bases in 1 I perchloric acid was allowed to
filter into the resin bed of a 1 by 27 cm. Dowex 50 column
in hydrogen form*

Elution with 100 ml. of water served to

remove uracil mixed with perchloric acid.

Cytosine came

off in an SO ml* volume after 160 ml. of 2 I hydrochloric
acid had been passed through the column*

Guanine was next

eluted by 3 M hydrochloric acid in a ISO ml* volume after
©0 ml* of the eluant had passed through the column, and
adenine was then removed by 140 ml, of 4 H hydrochloric
acid after a fore-run with 00 ml, of acid.
The uracil-perchloric acid solution was carefully
taken to 3 ml, volume and basified to a pi of 11 with © H
potassium hydroxide. After removal of the potassium per­
chlorate precipitate, the solution was placed on a 1 by 10
cm* Dowex 1 column in chloride form*

After the solution

had filtered into the resin bed, 100 ml. of 0.015 M
ammonium formate buffer of a pH of 9,1 was passed through,
and uracil was then removed in a 75 ml, volume after 75 ml.
of 0.016 M ammonium formate buffer of a pH of 8.0 had

31

filtered through the column.

It Is worthy of note that it

had been found that a slightly more aoldic buffer was re­
quired to obtain a sharp peak on elution of the slightly
more basic uracil than with thymine.

The difference In pKa

values is very small* the value for uracil being 9*46 and
that for thymine being 9 *88 * The effluent containing uracil
was evaporated to a small volume and placed on a 1 by 27 cm.
Dowex §0 column la hydrogen form for the removal of ammonium
ion*

The column was treated with 100 ml. of water which

served to wash through all the uracil* the ammonium ion
being retained on the resin*

The water solution of uracil

and formic acid was evaporated to dryness to remove the
latter* leaving a white residue of about 0.5 mg. of uracil.
Purification of Turin# and Pyrimidine Bases. Aliquots
of the chromatographic fractions' from the column separation
of each base were plated and counted as described for the
methionine-labeling studies* (see page 16) except that 1 ml.
fractions of all the bases were plated. The procedures for
quantitative determination of the bases by their absorption
characteristics were likewise the same as those used for the
previous study (see page 16)*
Recrystallization.

However*., the various fractions

proved to be grossly contaminated with other radioactive
compounds* and re chromatography on both cation and anionexchange resins proved of no avail in attempts at purifi­
cation*

The technique of recrystallizatlon to constant

32

specific activity was therefore tried* making us© of the
general insolubility of these amphoteric substances at
neutrality*

fhe adenine, guanine, and cytosine were each

evaporated to dryness several times to remove hydrochloric
acid, and then all four of the bases were taken up in 0*1 H
hydrochloric acid, transferred to 25 ml* volumetric flasks
and made up to volme with the same solvent*

the concentra­

tion of each solution was determined by measurement of its
optical density at the characteristic absorption wavelength,
and the compounds were then diluted with known amounts of
the unlabeled bases*

The pyrimidines were diluted with

9 parts of the unXabeled pyrimidine and the purines, be­
cause of their higher concentrations, were diluted with
19 parts of the unlabeled purines*

In most of the experi­

ments only cytosine and guanine were used, the other bases
being retained for future degradation studies*

fh© acid

solutions of the diluted compounds were then neutralised
with 0*1 V sodium,hydroxide, and then evaporated to 2 ml*
volume, and placed in the cold overnight*

fhe precipitate

was centrifuged off, taken up in 0*1 I hydrochloric acid,
the solution made up to 25 ml* volume, plated on platinum
dishes, and counted*

The procedure was repeated as many as

four times, but the specific activity did not approach a
constant value, and the ratio of optical densities at 260
and 280 m/i did not agree with the values determined for
pure samples*

However, the specific activity of the

38

pyrimidines generally increased with successive recrystalli­
sations toward a maximum value of 6,000 c«p.m./^iM, and the
Specific activity of the purines generally decreased with
successive recryetalligations toward a minimum value of
600 c.p *m*/^pM*
paper ehromatograbhy* Success was
finally obtained In purification of the purine and pyrimi­
dine bases by the use of a one-dlmens1onal paper chromato­
graphic procedure*

A solution of each base in 0.1 M

hydrochloric acid was applied in a narrow band 3*5 inches
fro® one end of a 7’by If "strip of Whatman Mo* 1 filter
paper*

Careful application of a narrow band was possible

using a 100 pi* micropipet attached' to a hyperdemle .
syringe*

Approximately 75 pi of solution was applied at k

time* the papers being s Wretched between books and aired, by
a fan to promote rapid drying*

After complete application

of the solutions, the papers were folded 1 inch from the
sample end and then in the opposite direction 2.25 inches
from the same end*

The 1-ineh flaps of the paper strips

were then placed in glass troughs and the papers passed up
and over glass rods (each of Which was above and to the
side of a trough,parallel to it) such that the second folds
coincided with the glass rods and the papers therefore hung
straight down fro® this point*

The two troughs were held

at the top of an all-glass rack, and the entire system was
contained in a 12 by 24 inch battery jar.

The solvent used,

54

* mixture of 2- propanol, water ana hydrochloric acid, was
that described toy Wyatt (59) for the separation of the purine
and pyrimidine bases of the nucleic acids*

In this solvent,

the Rf values (the Rf value toeing defined as the ratio of
13a# distance of migration of the compound being chromato­
graphed to the distance traveled by the solvent) for the
various bases are sufficiently different to obtain separa­
tion la the event that the contaminants are of this nature,
and, in addition, the distances of migration are of inter­
mediate value, making possible separations from materials
which are either very soluble in the solvent, and would
therefore migrate with the solvent front,'or very insoluble
in the solvent, remaining at the origin during development*
The solvent was added to each trough in approximately SO ml.
amounts, and a 100 ml* portion was placed in the bottom of
the Jar, to aid in saturating the atmosphere*

The jar was

then covered with a glass plate and the solvent allowed to
flow down the paper until it had almost reached the bot­
tom, a process which required about 24 hours.

The papers

were then removed and dried, and viewed with ultraviolet
light from a Mln«yaHght# lamp* the so-called quenching
technique#

The pyrimidines and adenine appeared as dark

blue bands on a light blue background, and guanine, which
fluoresces ©lightly, appeared a© a light blue band on the

*tatra-Violet Products, Inc., South Pasadena,
California

35

blue background• The bands were out out and eluted by means
of attachment to a filter paper strip dipping Into 0*1 N
hydrochloric acid*

The acid traveled down the paper and

then through the band, and was collected In a 50 ml* beaker.
The eluate® were evaporated to drynee® and re chromatographed
by ioa-exdbaage chromatography * On determination of concen­
tration and radioactivity the fractions were found to be
pure, as judged by the graphing method.

(see oage 21)

ftpoaifio Activity Determination. 'In subsequent experi­
ments the bases were eluted from the paper chromatograms,
transferred to £5 ml* volumetric flasks, diluted with, 0*1
$ hydrochloric acid, and the concentration and radioactivity
determined#

The concentration was ascertained by measure­

ment of the optical density at the wavelength of maximum
absorption*

This value, when divided by the molar extinc­

tion coefficient for the appropriate compound in 0*1 M
hydrochloric add, give© the molar concentration * Radio­
activity was determined by plating 1 ml* aliquots of the
solutions and treating as outlined previously (see page 16).
Uracil and Cytosine had specific activities of about 5000
©.p.m./juM, whereas the values for the purines were found to
be about 900 c.p.m./pM.
Localisation of Isotope.

In an attempt to localise the

radiocarbon of the uracil molecule, the degradation procedure
of Heinrich and Wilson (1) was carried out with the diluted
compound.

In preliminary experiments 10 mg. of twice-

96

reerystaliized uraoil was dissolved in 5 ml* of hot, carbon
dloxide<*frse water in the bottom half of a weighing bottle
of about 50 ml* capacity*

the bottle wae then sealed with

a greased rubber stopper which held a pair of pH electrodes
connected to a portable Beckman model & pH meter*, a pair
of 10 ml* buretsf and two glass tubes*

One of the tubes

extended almost to the bottom ©f the bottle, and was con*
nested to a scrubbing tower containing 10 H sodium hydrox­
ide*

Air from a compressed air line was slowly bubbled

through the tower to be freed of carbon dioxide, and then
passed into the bottle, serving to agitate the contents
and also to carry air contained in the bottle out through
the other glass tube*

the outlet tube, which just barely

extended into the bottle, served to e arry the air to a
capillary which extended to the bottom of a 7-lneh test
tube containing saturated barium hydroxide solution*
After bubbling through this solution the air was allowed
to escape through an outlet in the stopper which covered
the absorption tube*

After about ten volumes of air had

passed through the reaction bottle, 0*4 1 potassium
permanganate was added dropwise by means of one of the
burettes, and 0*6 I sulfuri© acid was added through the
other burette to maintain a pH of 6, as long as the perman­
ganate was decolorized*

Carbon dioxide, presumably

^National feohnlcal Laboratories, S* Pasadena,
California

37

representing a mixture of carbon atoms 4 and 6 of the
degraded pyrimidine (40, 41) was swept over into the ab­
sorption tube and trapped as barium carbonate*

When no

more precipitate was formed, the absorption tube was re­
moved, the contents transferred to a 12 ml* centrifuge
tube and the precipitate centrifuged off*

The dried bari­

um carbonate weighed 16 mg*, or 60 per cent of the theo­
retical value*

The reaction bottle was also removed and

the contents treated with 3 per cent (v/v) hydrogen per­
oxide to remove excess permanganate*

The precipitate of

manganese dioxide was filtered off, washed with hot water
and the combined filtrate and washings transferred to a
SO ml* centrifuge tube and made strongly alkaline with S
drops of 10 per cent (w/w) sodium hydroxide*

After heat­

ing in a boiling water bath for 3 minutes to hydrolyse the
oxalurie acid, the tube was removed, the contents neutral­
ised to a pH of 6 with S per cent (v/v) acetic acid, and
calcium oxalate precipitated by the dropwise addition of
10 per cent (w/w) calcium chloride*

After adjusting the

pH of the solution to 7*5 with dilute ammonia, the mixture
was centrifuged, and the washed precipitate transferred to
the reaction bottle used previously and dissolved in 5 ml.
of g H sulfuric acid*

After attaching the bottle to the

system used for the uracil oxidation and sweeping out all
carbon dioxide, 0*4 H potassium permanganate was again
added until no further discolorisation occurred.

The carbai

58

liberated* representing an average of carbon a boos
4 and 5 and carbon a tome 5 and 6 of the original uracil* was
again trapped aa barium carbonate*

33a© yield was 20 mg,,

or SO per cent of the theoretical value*

Th© filtrate re­

maining from the precipitation of calcium oxalate was evap­
orated to 5 ml* volume* and a hydrolysis of the urea con­
tained in this- solution was carried out*

The solution was

placed in the reaction bottle and* after attaching the bottle
to the system and sweeping out all carbon dioxide* a suspen­
sion of 25 mg* of urease powder* in 5 ml* of water was added
through the outlet tube*

The mixture was warned in a 50°C.

water bath for 15 minutes * and the resulting ammonium car­
bonate was then d ©composed by the addition of 10 ml* of 2 N
sulfuric acid*

fhe liberated carbon dioxide was then trapped

as barium carbonate in the absorption tube*
Prior to degradation of the labeled uracil* the samples
from the various experiments were pooled* and the total
quantity determined as 0*93 mg. and the specific activity
as 2170 c.p.m./pE*

One-half of the sample was diluted 20

times with unlabeled uracil* and the resulting 9*3 mg* then
had a specific activity of 108 e*p»a»/pE*

This sample was

then placed in the reaction bottle and degraded to carbon
dioxide and oxaluric acid by the action of permanganate*
The resulting barium carbonate was transferred to a 12 ml.

*Armour and Company* Chicago* Illiaoi®

59

centrifuge tube, centrifuged and the barium hydroxide super­
natant removed*

the precipitate wee suspended in ID ml* of

absolute ethanol and 1 ml* aliquots were plated on platinum
dishes, evaporated over a heating lamp, and counted In the
flow counter*

The plate® were- then weighed and the specific

activity calculated#

The barium carbonate samples obtained

in the oxalate oxidation and the urea hydrolysis were treated
similarly*

Compensation for self-absorption by the precipi­

tate® was effected by platin'g the same amount of barium
carbonate for each step of the degradation*

The results

could thus be expressed as a percentage of the total
activity of the uracil molecule#
The entire procedure was repeated on a larger scale,
using 2$ mg# of uracil*

Th# other half of the labeled

uracil (0*465 g«) was diluted SO times by the addition of
92*3 mg* of ualabeled uracil, and the specific activity of
the diluted compound was then 45 e *p

One refine­

ment, that of placing the calcium oxalate in the reaction
vessel and flushing the system prior to dissolution in
sulfuric acid, was carried out to obviate the possibility
of isotope loss due to conversion of any side-produets to
carbon dioxide by the acid*

Two mg* samples of barium car­

bonate were again plated In all steps*
The results of th© uracil degradation are shown in
Table XV.

It can be seen that th© distribution of activity

calculated on the basis of th© utilization of carbon atoms 2,
3 and 4 of aminobutyric acid for the carbon chain of th©

40

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41

pyrimldin© does not agree with the pattern found•

It was

assw e d that carbon atoms 2, 4 and 6 of th© pyrimidine ring
would pose ess the same degree of random labeling as that
determined for the purines and the protein amino acids#

Th©

value determined for carbon 2 of the pyrimidine seems to be
in agreement with this assumption, but the label of the
aminobutyric acid appears to have been spread over carbons 4,
& and 6 of the pyrimidine rather than specifically incorporated
Into carbon 5*

These results suggest some type of cleavage

and recombination of the- precursor In such a way that two of
the carbon atom© of the chain become highly labeled#

the

possibilities also exist that either the uracil contained a
radioactive impurity Or that the degradation did not occur as
supposed, 1
-Study of the trichloroacetic Acid Extract

isolatiim of MM-soluhM I&oloslMM
acetic Acid Extract *

Mm

ZsSste&sr

It was of interest to study the solu­

tion obtained by extraction of the dried Meurosoora mycelia
with cold 10 per cent (w/v) trichloroacetic acid, since this
fraction was found to contain one-quarter of the total radio­
activity fixed• The solution was therefore placed in a
100 ml# separatory funnel and extracted 5 time© with ethyl
ether#

The trichloroacetic acid-free aqueous solution was

then evaporated to dryness, taken up in water, and a small
portion chromatographed by a two-dimensional paper chromotographic procedure suggested by the work of Sanger and Tuppy (42)

42

and of Partridge and Westall (43)«

Th© solvents used were

water-eaturated phenol and butanol - acetic acid - water*
The attempted separation of this total trichloroacetic
acid extract by the ascending technique was unsuccessful,
but the methods used, which were found very useful in
later experiments, are described below*
fwo*4isaenaiona 1 Chromatography*, Whatman So* 1 filter
paper sheets (18 by 22 Inches) were ruled 3*5 inches from
the edge on two adjacent sides, and the origin marked at
the point where the lines crossed*

The origin was spotted

with the sample while a fan was directed at the paper to
hasten drying*

In eases where unbuffered neutral solvents

were to be used for development, it w as found necessary
to neutralise acidic sample® by placing the still-moist
spot over a beaker of moderately concentrated an mania,
and covering the area with a watch glass * The samples
were applied in 10 pi* amounts by the use of micropipets
or glass tubes drawn out to a fine capillary*

After the

sample had been applied, the papers were attached by one
of the aon-ruled edges to 24-inch glass rods by means of
stainless steel clips*

The papers were then hung into

glass troughs, the bottom edge of each paper having been
folded to form a one-inch flap sfcleh lay in the trough.

A glass rod was placed on each flap, to supply tension and
prevent the papers from touching each other since two papers
were dipped into each trough.

The entire system was

43

contained in a Ghrematocab* type chromatography chest*
3h subsequent studies of the trichloroacetic acid
extract, a descending technique was found more convenient*
She papers were folded about 1*?S inches above the flap,
end the trough* were placed at the top of the cabinet,
with the papers dipping into the trough* and then passing
up and over 24-inch long glass rods and hanging down
almost to the bottom of the chest*
Chromatoc&b Model Bm

The cheat wa* a

which was provided with an inner

glass cover with small holes through which solvent could
be readily added to the troughs*
For reproducible results it was found advisable to
equilibrate the chest for 18 hour* prior to addition of
the solvent to the troughs•

This consisted of placing a

dish of the solvent - with or without additions such aa
potassium cyanide, ammonia, etc* ■«* in the bottom of the
sheet, and then preparing all the material* for addition
of the solvent*

The syetem was then closed and th© atsios-

phere allowed to become saturated for the 18-hour period*
The solvent was then added to the troughs either by
the use of a long tube which was passed in through a hole
near the bottom of the chest * in the ease of the ascending
setup * or was added by passing the outlet of a separatory

^Chromatography Division, University Apparatus Co*,
Berkeley, California
^Research Equipment Corporation, Oakland, California

44

through holes in the inner cover of the chest* as
in the descending technique*

The solvent gonerally required

about 24 hours to travel the length of the papers, at which
time they were removed and dried In a forced draft of cool
air*

After rinsing to remove excess solvent if necessary,

the papers were rotated through 90 degrees in order that
the second ruled side could be folded and dipped into the
troughs*

After equilibration, the second solvent was

added to the troughs and allowed to travel almost the full
length of the papers . The papers were then removed and
dried in a forced draft of cool- air as before, preparatory
to localisation of spots by ultraviolet quenching of by
spraying with the appropriate color reagents*
Xon-exohangse Cliromotojsraphy*

In subsequent experi­

ments the ether-extracted solution of the trichloroacetic
acid extract was basifled to a pH of 11 and placed on a
1 by 27 cm* column packed with Dowex 1 resin of 12 per
cent crosslinking and 100 to 200 mesh sis© which had been
previously converted to the chloride form*

The column was

eluted with 90 ml* of water, followed by 165 ml* of 2 H
•hydrochloric acid*

Fractions of 15 ml. volume were collect­

ed, and the ultraviolet-absorbing materials were found to
be eluted in the first 60 ml. of the 2 N hydrochloric
acid effluent*

The peak absorption of these compounds

was in the 260 mji range*

46

& « » g » H o a Si a tejMaS Nucleotide. Since It appeared
moat probable that the purine nucleotides would predominate
in this extract, it was decided to hydrolyse a portion with
1 M hydrochloric acid for one hour on the steam bath, a
procedure suggested by the work of Smith and Markham (44).
Since the pyrimidine nucleotides are much more stable to
apid hydrolysis than the purine nucleotides, the former
should be unaffected and the latter should appear as the
free purine bases, which are readily separated from nucleo­
tides in a variety of solvents,

Accordingly, half of the

residue obtained by evaporation of the 60 ml, volume con­
taining the nucleotides was taken up in 8 ml. of 1 K
hydrochloric acid and placed in a glass-stoppered 2 ml*
volumetric flask.

The mixture was heated, on the s team

bath for 1 hour, then evaporated to dryness to remove
hydrochloric acid and both hydrolysed and unhydrolyzed
mixtures spotted on Whatman No* 1 sheets to be developed
by the two-dimensional descending technique.

The solvent

used for the first direction was 2- propanol-hydrochloric
acid - water, as described by Wyatt (59), and the s eeond
solvent was butanol - water - so mania, as described by
*

Hotchkiss (46) and applied to nucleotide separations by
Wyatt (46),

By ultraviolet quenching two dark blue areas

were discernible in the chromatogram of the hydrolysed
extract, one of Rf *9 and the other of Rf *7 in is©pro­
panol-hydrochloric acid*

Both were located on th© base

46

1184 of th* butanol - water * ammonia development, and
hence could be nucleotides, since tlies© substances do
not migrate in the latter solvent*

Spots were also dis­

cernible at -Rf values corresponding to adenine and guanosine, and several very faint spots were detected in the
area to which free pyrimidines and their nucleosides would
migrate#

The two spots containing possible nucleotides

were out out and eluted with 0*1 n hydrochloric acid*
adjacent areas were also cut out and eluted with 0*1 $
hydrochloric acid to s erve as blanks for the determina­
tion of concentration*

The ultraviolet absorption curve

as recorded by the Beckman Ultraviolet Recording spectro­
photometer* indicated a peak absorption at £64 mp for the
material of Rf *? in 2— -propanol - hydrochloric acid, and
indicated only end-absorbing material for the spot of
Rf *9*

fia contrast, the chromatogram for the uhhy&rolyzed

extract yielded four possible nucleotide spots at Rf
values of *5, .4, *76, and *9 in isopropanol - hydrochloric
acid, and the spot for adenine was of considerably de­
creased size*

These results, coupled with approximate

agreement of the Bf value with that of a known uridylle
acid sample, strongly suggested that the nucleotide of
Rf *7 was a uridine nucleotide*

Since data was avail­

able on the behavior of th© 5* -nucleotides of uridine.

National Technical Laboratories, S. Pasadena,
California

47

cytidine, and adenosine (47) in isobutyric acid - ammonia
{the values being *23,| «Sdy and .45 respectively), and
because M&gas&nlck et jgl* (48) had studied the nucleic
acid nucleotides in this solvent* it was decided to apply
one-dimenaional s trip chromatography* as described pre­
viously (see page 53)* using this solvent*

the nucleo­

tide material of If #7© from the two-dimensional chromat­
ogram of the unhydrolyaed extract was eluted with 0*1 N
hydrochloric acid* the eluate was evaporated to dryness*
and taken up in a small amount of 0*1 H hydrochloric
acid*

The solutions of the pres used uridine nucleotides

were then spotted 8*23 inches in from the edge of the 7inch paper strips* and known uridine* uridylic acid and
adenylic acid were spotted 2*25 inches In from the
opposite edge*

Uridine-5* -phosphate was not available

for chromatography at this time*

The papers were de­

veloped until the solvent had almost r cached the end of
the strips and then removed and dried in a hood*

By

the ultraviolet quenching technique spots were discernible
at Rf *18 for the unhydrolyaed nucleotide, and at Rf *19
for the hydrolysed nucleotide*

These compared with a

published value of *23 for uridine-5* -phosphate, which
appeared to be rather good agreement since the known com­
pounds were found to have Rf values also slightly less
than those in the literature.

Other ultraviolet-absorbing

areas were found at Rf values of *62 and *66.

The various

spots were cut out and ©luted with 0*1 » hydrochloric acid.

48

Bit eluatea were made up to 25 ml. and the optical densities determined for each over the range from 236 to 280 mju.
$he compounds of Rf *18 and *19 were the only materials
showing the typical nucleotide absorption curve, th© peak
in both oases being at 262 mju, the raa.vij©inm absorption
characteristic of uridine nucleotides*

Adjacent areas

for these spots were out out and ©luted with 0*1 N
hydrochloric acid to serve as blanks for comparison, and
the optical density at this wavelength was used to deter­
mine the concentration of the compound, using the molar
extinction coefficient for urldin©-5' -phosphate.

On© mi.

aliquots of the various solutions were also plated and
c o m ted as described previously (see page 16).

'Eh©

specific activity was found to be 4000 c*p.m./pM*
Characterization of Uridlne-S1-phosphate* An attempt
was also mad© to definitely establish the position of the
phosphate group in this uridine nucleotide by means of the
phosphate^liber&tiag action of the specific 5* nucleoti­
dase of rattlesnake venom* described by Gulland and
Jackson (49).

A sample of this crystalline venom was

kindly supplied by Dr* H* A# Llllevik.

Eight ml* of the

solution of the nucleotide of Rf .18, containing 0*4 pM,
or approximately 0*36 rag., was evaporated to dryness and
taken up in 0*8 ml* of a sodium carbonate solution

% © « » Allen Reptile Institute, Silver Springs, Florida

49

ooataining 0*1 sal* of half-saturated sodium carbonate
solution*

A known sample of 0*5 mg, of uridine-S*-

phosphate was treated similarly,

Five mg, of snake venom

was added to each sample In a 5 ml, glass-stoppered volu­
metric flask, and this was followed by addition of 3 ml,
of 0*1 M sodium borate buffer of a pH of 8,7,

Chloroform

was then added to each flask in 230 pi, amounts, and the
flasks were stoppered and incubated at 37°G for 3 hours*
fhe mixture® were then evaporated to dryness in vacuo and
the residues transferred to 12 ml* centrifuge tubes with
the aid of 0*1 M hydrochloric acid*

After centrifugation,

the supernatants for known and unknown were spotted on
separate Whatman No* 1 strips and developed with isobutyric
acid * ammonia solvent*

Known uridine-5 ♦-phosphate and

uridine samples were also chromatographed with and with­
out added borate buffer*

the latter had been concentrated

to simulate the salt concentration of the ensyme-treated
samples,

‘The developed chromatograms, after drying, were

found by ultraviolet quenching to contain ultravioletabsorbing material only near the solvent front, suggesting
that complete conversion to bases had somehow occurred.
The poor definition of the spots on these chromatograms
prompted a decision to repeat the process with the other
pyrimidine nucleotide sample, of 8f ,10*

The results with

the developed papers from this experiment w ere again in­
conclusive in the case of the unknown, (there being a

50

fluereaeent spot at Rf *18 but no nucleoside spot) but
were conclusive in establishing the activity of the enzyme
and the validity of the procedure since the known uridine5* -phosphate gave a strong spot at the Rf value for
uridine and .a faint spot for the residual, unhydrolyzed
nucleotide*

The known Rf values to which these results

were compared were those which had been determined by
addition of concentrated borate buffer to known uridine
and uridlne-5*-phosphate prior to chromatography •

It

seemed possible that some impurity in the isolated nu­
cleotide might be either disrupting the normal course of
reaction with snake venom 5’-nucleotidase or radically
altering the rate of migration of the products*
Therefore, th® entire isolation procedure was re­
peated, using a fresh trichloroacetic acid extract ob­
tained from the mycelial growth from 20 flasks of the
aminobutyrlc acid-fed mutant, as described in an earlier
section (see pages 41 to 44).

Two-dimensional paper

f t h togrephy yielded ultraviolet—absorbing spots of
nucleotide character at Rf values .54 and .6 in isopro­
panol-hydrochloric acid*

A fraction of each was again

hydrolysed, and the various portions reohromatographed
on vvhatman So* 1 strips in isobutyric acid - snmonia.
After drying the developed chromatograms, they were view­
ed by ultraviolet quenching and the spot of Rf *18 again
toxoid In each case*

The nucleotides were then removed

51

with 0.1 M hydrochlor1c acid and placed on other Whatman
K©* 1 stripe for chromatography in tertiary butanol hydrochloric acid - water, a solvent described by Smith
and Markham (44).

After development, spots were found

at Rf values *56 and *76, the latter value comparing with
a spot for known uridine-5*-phosphate.

After elution, the

material of the lower Rf value, which also contained a
fluorescent fraction, was end-absorbing when studied ae
to ultraviolet absorption over the £36 to 280 mja range*
the nucleotide of Rf .76 read versus a blank obtained by
elution of areas surrounding the spot showed a peak opti­
cal density reading of *360 at 262 mp, and the typical ab­
sorption curve of uridine-S*-phosphate*

Aliquots of the

eluate were also plated and counted as described earlier
(see page 16} • The specific activity was found to be
1400 c*p*is./jaM.

The hydrolysis of the 5*-phosphate group

by means of snake venom carried out with this purified
nucleotide gave a faint spot for uridine on subsequent
rechromatography in the same solvent.

On the basis of

chromatograph!o behavior, ultraviolet absorption and 5*
nucleotidase treatment, it was therefore concluded that
the unknown pyrimidine nucleotide was urldlne-5*-phosphate •
Separation of an Adenine Hucleotlde* Small portions
of the original hydrolysed and unhydrolyzed, trichloro­
acetic acid-free extracts were also banded on Whatman Ko.l
strips and chromatographed in butanol - formic acid « water,
a solvent described by Markham and Smith (60).

After

58

development, ultraviolet quenching Indicated a band at
the origin which apparently consisted of nucleotides,
since these compounds do not migrate in this solvent *
Bands were also detected at Hf *08,

which probably wasdue

to action between hydrochloric acid

and the

paper, andat

Rf #15 and *18, which was most probably a purine base or
nucleoside band*

The pattern was essentially the same

for both hydrolysed and unhydrolysed samples*
To establish that the bands at the origin were in­
deed nucleotides, the phosphorus determination of Hanes
and ISherwood (51) was applied

to a

chromatogram.

sprayed at a rate of 1 ml.

The strips were

portion

of each

per 100 sq# cm*, with a solution containing 5 ml* 60 per
cent perchloric acid, 10 ml* 1 M hydrochloric acid, 85 ml.
of 4 per cent (w/v) ammonium molybdate, and water to make
100 ml*

The papers were then heated to 85°C in a chroma­

tography oven for 7 minute®, and finally placed in a
graduated cylinder containing hydrogen sulfide.

Unfor­

tunately, the entire paper turned black suggesting that
metal ions, especially lead or copper, were present in
the paper*

An attempt was therefore made to use a stan­

nous chloride spray for reduction of the phoaphomolybdate
complex, but the entire strip turned blue, making differ­
entiation of the nucleotide band impossible*

Apparently

acid-washed papers were needed for a successful phosphorus
analysis*

63

Character!zatten of Adenoslne-5* -phosphate. The
nucleotide bend from the remaining portion of eaeh chroma­
togram m e therefore out out and eluted with 0.1 N hydro­
chloric acid*

The eluatee were taken to dryness, the com­

pounds banded on Whatman $o • 1 strips, and the chromato­
grams developed with tertiary butanol-hydrochloric acid water (44)#

After development, there appeared only one

ultraviolet-absorbing band, at Bf »&., which corresponded
to a purine nucleotide*

lb the chromatogram of the

nucleotide fraction which had been hydrolysed, the band
was very much attenuated, compatible with the expected
destruction of purine nucleotides under these conditions,
Furthermore, the fact that this compound appeared to possess
some degree of acid stability suggested that it was a 5*nucleotide, since Ochoa (52) has demonstrated the much
greater acid stability of the purine-6*-nucleotide® under
these hydrolytic conditions compared to purine nucleotides
having phosphate attached at the 2* and 3* positions of the
sugar moiety*

Its ultraviolet absorption spectrum in 0.1

N hydrochloric acid showed a maximum at 255 rap, and other­
wise agreed exactly with the absorption curve of the
adenine mononucleotides♦ From these several lines of
evidence the conclusion was therefore reached that the iso­
lated nucleotide was adenosine-5*-phosphate•

Concentration

and radioactivity measurements were carried out in th© same
manner as with the isolated uridine-5*-phosphate.

The

54

specific activity was found to be 800 c*p*ra*/;iM*
A parallel study was made of the nucleotides of the
trichloroacetic acid extract after growing the mutant
strain on unlabeled uridine rather than labeled amino—
butyric acid*

She pattern of nucleotides and bases showed

no radical differences*
Eadloautograohy of Paper Chromatoarama * Sheets and
strips to be radioautographed were taped to 14 by 17 Inch
sheets of Kodak Blue Brand X-ray film**

It was found

helpful to place several spots of a radioactive solution
at marked positions on the papers to serve as a key in
order that the developed films and the corresponding
chromatograms could later be correctly matched*

fhe

films, with attached papers, were placed between plywood
boards which were then clamped tightly together to insure
close contact of chromatograms and films*

Pieces of thin

cardboard were found sufficient as separators between the
various radioautograms• After a period of 3 weeks the
chromatograms were removed and the films were developed
for four minute® with Kodak D-19 developer, then placed
in a stop bath of 1 per cent (w/v) acetic acid for 10
seconds, and left in a fixer solution of sodium thiosulfate for at least 10 minutes * $he films were then
washed in cold tap water for approximately 1 hour, and

*E&stman Kodak Company, Hocheater, Hew York

55

th#» h w g by ©lips t© dry*

An amber Kodak Safelight,

Series 6-B, was found useful for the darkroom procedures*
Isolation of Amino A©ids from the Trlchloroacetlc Acid
Duplicates of th© butanol - formic acid - water
chromatograms of the trichloroacetic acid extracts of the
mold which were prepared in studies of th© acid-soluble
nucleotides (see page 51) were sprayed with 0,02 per cent
(w/v) ninhydrin In water-saturated butanol*

Several nin-

hydrin-positiv© spots were obtained, one of which had the
Rf value *27* the value for alpha-aminobutyrlc acid in
this solvent*

Since this spot was present in similar

amounts in chromatograms of both the hydrolyzed and non­
hydrolyzed extracts, it was probably not a peptide*

This

material was eluted from th© paper and its concentration
and radioactivity determined• The high specific activity
suggested that this might indeed be the precursor, aminobutyric acid* and purification of this compound was there­
fore undertaken*

On chromatography in both ohenol - water -

cupron and la butanol - water - ammonia, th© Rf value of
the compound compared precisely with known alpha-aminobutyric acid*

The constancy of the specific activities of

the eluates of the spots at each stage of purification
indicated that the compound was essentially radlochemi©ally
pure after the final chromatography.

The specific activity

determined from the final measurements of concentration and
radioactivity was found to be 2500 c*p.m./hM*

Other

56

radioactive spots of the acid-soluble pool were tentatively
identified by chromatographic procedures as isoleucine,
methionine sulfone, and beta-alanine .
An investigation of the occurrence and quantity of
the amino acids of the acid-soluble pool of the wild strain,
grown without alpha-amlnobutyric acid, was also carried out*
Chromatography on long strips of Whatman Ho. 5 paper gave
a separation of eight ninhydrin-poei tive spots, one of
whleh coincided with a known spot of alpha-aminobutyric
acid*

The normal mold thus appears to possess an amino-

butyric acid pool*
The smeller else of the spot for aminobutyric acid
of the wild strain as compared to the corresponding spot
for the mutant suggests that the pool size of this amino
acid is normally small, but that a relatively large pool
exists in the mutant when grown on aminobutyric acid*
Study of the Amino Acids
of the Soluble Proteins
Isolation and Hydrolysis of the Soluble Proteins. The
alkaline hydrolysis treatment for the separation of ribo­
nucleotides (see page 28) also served to extract the
soluble proteins from the mycelia. The precipitate ob­
tained by acidification of this extract with concentrated
perchloric acid in the cold therefore contained protein®,
a® well a® potassium perchlorate and some polysaccharides*
The proteins and polysaccharides were redissolved by

57

stirring the precipitate with X H sodium hydroxide for 12
hours*

The residual potassium perchlorate was centrifuged

off* and the dissolved materials reprecipitated with 1 If
hydrochloric acid*

On attempting to dry the protein by

means of an ethanol wash, the major portion of the mix**
tore dissolved, leaving a small amount of a light brown
gelatinous precipitate*

The latter was centrifuged down,

and the supernatant was evaporated to dryness, yielding a
idilte, amorphous precipitate weighing 76 mg*, and pro*
sumably consisting mainly of alcohol-soluble protein*
This precipitate was next transferred to a 12 ml*
centrifuge tube and hydrolyzed with 4 ml* of 6 If hydro­
chloric acid on the steam bath for 24 hours to bring about
hydrolysis of the protein to the constituent amino acids*
Xfcte mixture was then filtered, and the solution was evap­
orated to dryness several time® to remove hydrochloric
acid*

The residue was taken up in water, transferred to

a 12 ml* centrifuge tube, and evaporated to 0*5 ml. for
chromotography*
Separation and Purification of Protein Amino Acids*
Preliminary experiment®, carried out with the hydrolysate
from an unlabeled protein sample, demonstrated that a twodimensional paper chromatographic technique was capable of
resolving the amino acids of the crude hydrolysate without
prior purification on ion exchange resins.

The technique

which was used followed very closely hie procedure outlined

58

f©r tb# Sipftpation of th® acid-soluble nucleotides (see
pegs 42)#

The solvents used were phenol - water and

butanol - acetic acid - water, both of which were described
by ganger and Tuppy (42)«

it Is Important to note that the

hydrolysate had to be neutralised with ammonia# as mentioned
previously # when using an unbuffered neutral solvent suoh
as phenol - water# to prevent the streaking of the acidic
amine acids*

The equilibrating liquid consisted of 0*5 g.

potassium cyanide and 20*8 ml* of concentrated anmonia in
sufficient water to make 2 liters*

After development in

the phenol - water solvent# using the ascending technique,
the sheets were rmoved# dried# and then rinsed with an­
hydrous ethyl ether to remove residual phenol*

The sheets#

while still ellpped to the glass rods# w ere hung over a
sink and rinsed with ether fro® a wash bottle# while a fan
carried the vapors to a nearby hood.

After the papers had

dried# the bottom 8 inches of the papers were removed# and
the chromatograms were the® turned through 90 degrees and
equilibrated for the second solvent*

The equilibrating

solution in this case was the lower layer from the butanol acetic acid - water mixture.

After 18 hours of equilibra­

tion# the solvent was added and the papers developed for
the usual 24-hour period*

After drying in an oven at

48PG for 5 hours In a forced draft# the papers were
developed with a 0*02 per cent (w/v) solution of ninhydrin in water-saturated butanol.

The solution was sprayed

on the paper in an even film - but not enough to run -

59

wM1*

papers hung, with the origin at the top, in a

hood*

The papers were then heated to 85®C* in a chromat­

ography oven* and spots for a variety of amino acids be­
came evident in 15 minutes*
were tried*

Several amounts of hydrolysate

The heavier samples produced somewhat diffuse

and elongated spots* but the lower concentrations gave
reasonably clear separations* especially of the neutral
and acidic amino acids*

The concentration of proline

appeared quite high* and corroborated a suspicion* prompted
by the alcohol solubility* that the predominant basesoluble protein® of the Heurosnora mycelia are prolamine
in nature*

Clear spots were also obtained for the iso-

leucine - leucine mixture* for aspartic and glutamic acids*
and for valine*

Spots for serine* glycine and threonine

were somewhat diffuse#

Radioautograms of these chromato­

grams* made a® described in the previous section {see page 54}
indicated high labeling of the isoleucine - leucine mixtire*
some labeling of the proline fraction - probably explained
by Its hi^i concentration - and of the glutamic acid* but
little activity in aspartic acid or in th© serine threonine - glycine mixture*
A long-strip paper chromatographic procedure was also
found useful for the separation of the amino acids of th©
protein hydrolysate*

The hydrolysate was first placed on

a 2 by 50 cm* Dowex 50 column, which was then washed with
water* and th© amino acids then removed by elution with

60

9 ,IT hydrochloric acid*

The ©luafce was evaporated to dry-

ness to remove hydrochloric acid, preparatory to paper
chromatography«

The 42-inch long papers were prepared as

outlined for the paper strip chromatography used in connec­
tion with the studies of'the purine and pyrimidine bases
(see page 99).«

Heaty Whatman Ho* S paper was available*

and* in con junction with the banding technique* enabled
the separation of much larger amounts of material*

The

long paper strip® were dipped into troughs at the top of
a double-length rack which stood in a 12 by 24-ineh
battery jar*

A second jar* 1£ by 12 inches* was inverted

over the first after the solvent had been added* and the
point of joining was then sealed with mashing tape* The
solvent in the troughs usually had to be replenished once
in the course of development*

This was accomplished by

breaking the seal between the jars and raising the upper
jar sufficiently to attach a funnel of solvent to tubes
extending up to each trough*

The solvent found most use­

ful was butanol - formic acid - water (SO) used earlier
in connection with nucleotide separations*

On d evelopment

and comparison with known compounds* it was found that the
basic amino acids* which were of low concentration, remained
at the origin*

Proceeding out from the origin* aspartic

acid was obtained in a relatively well-separated band led
by glutamic acid mixed with serine and glycine.

A band of

threonine was next* followed by a wide, heavy band of proline, yellow In color, and then, after a gap of several Rf

61

unit®# a wide band of valine and a wider band of mixed
leucine — isoleucine« The valine and isoleucine — leucine
band® were well separated*
The spot® for the leucine - isoleucine mixture and
for both aspartic and glutamic acids from the two-dimen­
sional chromatogram® used for the separation of the hy­
drolysate of the first isolated proteins were out out and
eluted with water*
The leucine - isoleucine mixture was treated by the
method of Stein and Moore using catlonwexchange chromato­
graphy (63)*

fhe method* though unsuccessful for the

separation of the labeled mixture* was successful in
separating a known sample of 1 mg* each of leucine and
isoleucine*

fhe following modification of the method

is therefore presented because of its ease and simplicity*
A 0*9 by 100 cm* column was completely jacketed with a 3 cm*
wide glass tube which was in turn wound with a long heating
tape*

A variable resistance attached to the tape completed

a simplified apparatus for control of the temperature of
the column#

This was found quite capable of maintaining

the required temperature to within half a degree in the
course of the separation*

The column was packed with

Dowex 60 cation-exchange resin of IS per cent eros si ink­
ing and 100 to 200 mesh particle size by forming a slurry
of the resin - previously placed in the sodium form by
washing ia 1 1 sodium hydroxide and then in water - in 0*1 M

62

sodium citrate buffer of a pH of 5*41 and pouring this
mixture into th© column with gentle suction.

A mixture

of 1 mg* each of leucine and isoleucin© in 1 ml. 0.1 H
hydrochloric acid was mixed with 2 ml. of the citrate
buffer to give a final pH of 2*5 to 5, and the amino acids
were then allowed to filter into th© column*

.Elution wae

begun at the rate of 4 ml. per hour with the same citrate
buffer after the column temperature had been adjusted to
57°<3*

This alow rate of elution wae controlled by a

stopcock attached to the column outlet.

After 215 ml. of

the eluant had passed through the column, eluticn with 0*1
II sodium citrate buffer of a pH of 4.26 was begun, and the
temperature was increased to 50°G * Two nlnhydrin-pcsitive
peaks, as located by the quantitative determination of
amino acids suggested by Moor© and Stein (54) (see page 64),
appeared after 55 fractions of 2 ml. each had been collected,
using an automatic fraction collector*

On© amino acid-free

tub© occurred between the peaks, so th© procedure was deemed
useful for the separation of the labeled mixture of leucine
and isoleucine*

It is important to not© that the buffers

had to be made up with boiled water just prior to use in
order to avoid bubble formation on passage into th© heated
column.

The column was then repacked, fresh buffers were

made, the labeled mixture was acidified and filtered
into the resin, and elution with the first solvent begun. How­
ever, in the course of the night th© ©Huant channeled through the
stopcock and the resulting high flow rat© apparently spread

68

til# Amino acids through tho column • fh© mixture could not
be located, but a second labeled sample, obtained by the
strip chromatographic procedure, was successfully separated by paper chromatography*
*131# leucine - isoleueine band from the butanol formic acid - water s eparation of the protein hydrolysate
mas located by the ninhydrin method and was then cut out
and eluted with water,.

The solution was evaporated to

0*6 ml*, banded on the 4S-ineh long ifhatman Ho* 3 strips,
and chromatographed in the double battery jar apparatus
described for strip chromatography of the protein hydroly­
sate, (see page 69)

The solvent chosen for the separation

of these very similar amino acids was water-saturated
butanol, (88), in which leucine has an Ef value of *46
and isoleueine a value of *41*

The end of the papers had

been out to form a sawtooth edge and the solvent wae
allowed to drip from the end of the strips, thereby in­
creasing the distance of travel of the bands and aiding
the separation*

The bottom of the tank was layered with

a Bolution of the solvent containing 8 per cent (w/v) ammon­
ia*

After development the papers were dried and treated

with ninhydrin*

The bands of leucine and isoleueine were

clearly defined, though not completely separated*

There­

fore, the trailing edge of the isoleueine band and the
leading edge of the 3® ucine band were cut out and eluted
with water*

The eluates were made up to 25 ml, and 0,1 ml.

64

samples taken for Quantitative ninhydrin determination by
the method, of Moor® mad S M n

(64),

B m samples, and iso-

leucine standard® as well, were each mixed with 1 ml. of
a ninhydrin reagent consisting of 0,8 g« of stannous
chloride, 500 ml. of 0,1 M eodium citrate buffer of a pH
of 8, 20 g. of ninhydrin, and 600 ml, of methyl cellosolve.
lb# reagent was stored under nitrogen in the dark*

The

mixtures of samples and reagent, in 4-ineh test tubes,
were heated for 80 minutes in a boiling water bath and
then transferred to the ©ell® of a lil©tt-Summers on* color**
imeter and diluted with 5 ml, of a 1 to 1 (v/v) mixture of
water and n~prop&nal, The absorption of light, using a
green filter {670 mp), was measured for each sample versus
a blank of distilled water and reagent which had been
carried throw#* the entire procedure.

The absorption

values of the known isoleueine samples were plotted
against concentration to obtain a standard curve from which
the concentration of the Isolated amino acids could be de­
termined*

Aliquots of the eluates were also plated and

counted as described earlier (see page 16).

Isoleueine

was found to have a specific activity of 7000 c*p.m./pM,
whereas leucine was labeled to the extent of 1S00
The remainder of each eluate was evaporated to dryness and
rechromatographed with butanol — formic acid — water * The

*Klett Manufacturing Company, New York

66

quantitative d@ terminst1on and radioactivity measuneatents
repeated fop the elu&tes from these ohrome ■hftgr»om« t
In addition, radioautographs were made of duplicates of
the original leucine - isoleueine separation made with
butanol - water*

& portion of the isoleueine sample was

alee chromatographed with phenol - water - eupron, a
solvent consisting of water»saturat«d phenol containing
0*1 per cent (w/v) alpha * bensoinoxlme (eupron) (66),
and a second portion was mixed with the known compound,
chromatographed in ethanol - ether - water - axanonla (66)
and radioautographed#

2he specific activity of isoleueine

from, the chromatography in phenol was found to be 1650
$he identity of the Isolated compound with isoleucine .was proved by migration of the mixture as one ninhydrin-positive spot which appeared as the only radioactive
area on radioautography•
aspartic and glutamic acids were obtained, from twodimensional chromatograms and also from strip chromato­
grams of the protein hydrolysates*

2he samples w ere banded

on ihatman Ho# 5 strips and chromatographed in phenol water <* eupron*

Concentration and radioactivity'were de**

temlned as before#

Aspartic acid had a specific activity

of 300 e*p*nu/pH, and glutamic acid has a specific activity
of 550
Valine, obtained from the earn© sources, was treated
similarly, but with an added purification by rechromato-

66

gr&phy la butanol * water In a 3 per cent (w/v) ammonia
atmosphere• The specific activity of this amino acid

was 200 c«'p«n*/pai,
Threonine from long strip chromatograms was purified
by strip chromatography in phenol • water «• cupron and
It® concentration and radioactivity likewise determined*
The specific activity was determined as 300 c.p.m./pM.
Methionine was not located in the protein hydroly­
sate*
The results of the specific activity determinations
for the various isolated compounds are summarized in
Tables V and VI*

Table V contains the values found for

the compound® isolated from the leurospora mycelium after
the mold had been grown for 4 days on 0l^*labeled aLpha-

ftsAftobtttyrte acid* Table VI contains the specific -activity
values determined for compound® isolated from the mold
mycelium after 6 days growth.

The distinction is nee*

eessary since a considerable dilution of the labeled com­
pound occurred In the longer growth period* and result®
were cons idered comparable only if the time of growth of
the mold myeelia from which the compounds were isolated
was the same*
It can be seen that the am in©butyric acid is indeed
a pyrimidine precursor since a selective incorporation
into the pyrimidine occur®* though, the label is diluted
10 times In the course of growth.

The pattern also

67

t a b u :v

GARBON-14 CQJfTEHT OF CQMFQIMDS OF HEURQ5P0RA
AFTER 4 BATS GROWTH IH THE PRlfOpfEl
OF AMIM0BOTTRZC ACIiH^C*^
Specific Activity
(c *p*«u/pM*)

Compound

Mutant
Strain

Wild
Strain

Hueleic Acid Components
Adenine

850

Guanine

900

Brae 11

4300

Cytosine

5000

600

1650

nucleotides of Acid-Soluble Fraction
Adenosine-51-Fhoephate
Brldlne-5*-Phosphate

800
4000

Amino Acids of the Soluble Protein#
Aspartic Acid

500

Glutamic Acid

550

Threonine

300

-t

Leucine

1000

Isoleueine

5000

Amlnobutyric Acid Supplied

43000

48000

68

tmm

vx

CARBQH~14 COROTT OF COMPOUNDS OF NEUROSPORA 1298
AFTER 6 DAIS GROWTH IK T H B ^ S S l i M ^ O F
AMIJSrOBUTIRIC ACID-6-C14
.,...

iSotopouni

.... .

Nucleic Acid Component#

..

Activity

.

c*p.m./pM

Adenine

1000

Uracil

1700

Acid~Solubl© Fraction
Uridine-b* -phosphate

1400

Amlnobutyric Acid

2300

Amino Acids of the Soluble Protein#
Aspartic Acid

350

Glutamic Acid

650

Threonine

500

Valine

200

Isoleueine
Aminobutyrie Acid Supplied

1650
48000

ftppd&?B sImilar in th$ acid«*soluble nucleotide fF&ction«
Purth®naor©§ the route of utilization appears to be a
normal one* sine® the pyrimidines of the wild strain are
also labeled*
The labeling of the isoleueine is not unexpected
sineo Adslberg, Coughlin and Barratt (87) have shorn that

69

amlnobutyrie &oi4 is ui Intermediate In isoleueine bio**
synthesis in leuroapera. Tbs low labsling of valine also
lands support to the suggestion by these authors that
amlnobutyrie sold is not involved in valine biosynthesis*
The low labeling of aspartic acid precludes the possibility
of its involvement as such in the conversion of aminobutyric acid to pyrimidines*
the finding that amlnobutyrie acid of a specific
activity comparable to the pyrimidines is present in the
acid-soluble fraction of Hoursseer* explains the observed
dilution of the precursor on conversion to pyrimidine*

It

appears that once growth begins, the mold can then make
additional amlnobutyrie acid, and this endogenous acid
dilutes the precursor and therefore also the pyrimidines*
Study of the Final Nutrient Media
The nutrient medium remaining after growth of the
mold on labeled amlnobutyrie acid was evaporated to dry­
ness after the addition of oaprylic alcohol as an antifoaming agent*

That part of the residue which would dis­

solve in 0*1 1 hydrochloric acid was transferred to a I by
m

cm* column containing Bowex SO resin in hydrogen form.

The column was eluted with water and then 8 B hydrochloric
acid, and the eluates were evaporated to dryne©e» taken up
in 0*1 K hydrochloric acid, and chromatographed on paper
strips in butanol - formic acid - water.

The chromatogram

of the hydrochloric acid elmte showed no amlnobutyrie acid,

70

tout ninhydrin-posi tlve spots occurred at Rf values of .42
and *74*

These spots were shown to to# strongly radioactive

eaa sutoseluent radioautography*

The water eluate, which

protoably consisted largely of sugars, did not resolve on
chromatography *
Study of the Saponifiable Lipid Fraction
The ethanol*#ther extract of the mycelium was taken
to dryness in a 100 ml* round-bo tfcomed flask preparatory
to saponification toy the method of Weygand (58).

It was

estimated that the maximum amount of saponifiable lipids
in 1 g* of mycelium was 10© mg., mid the residue was
therefore treated with 3 times the theoretical amount of
alcoholic potassium hydroxide* or @0 mg* of the base.
& reflux condenser was connected and the mixture was re­
fluxed for one hour* at which time the contents of the
flask were transferred to a 1©0 ml. separatory funnel
with the aid of a small amount of ether, and extracted
with water#

The water extract was acidified with hydro­

chloric acid, then ether extracted, and the ether extract
made up to a 200 ml. volume in a glass-stoppered volumetric
flask*

A 1 ml. aliquot was diluted to 25 ml. and 1 ml.

aliquots of this diluted sample plated for counting.

The

remaining ether solution was evaporated to dryness and the
weight of the remaining fatty acids determined.

The pro­

cedure was carried out for the ethanol-ether extract of
the mycelium of tooth the mutant and the wild strains after

71

growth of eaeh on labeled aminobutyrie add*

The total

weight of saponifiable lipids In the ease of the mutant
was found to be 0*2 mg,, and the Bpecific activity was
determined using this value In conjunction with the
moleeular weight of palmitic acid#

The latter wae

assumed to approximate the average molecular weight of
the fatty acids of the natural fats of this organism#
Study of Sfeuroepova Deoxyribonucleic Acid
An attempt was made to isolate deoxyribonuoleic acid
with a view toward the study of the pyrimidine labeling in
this fraction.

It was considered probable that the basic

hydrolysis step for the liberation of the riboauclootidea
dissolved not only the soluble proteins but also deoxyribonucleic acid#

Several attempts were therefore made

to extract the latter compound from the precipitate of
proteins and polysaccharides obtained after removal of
potassium perchlorate (see page 28) # Extraction with
10 per cent (w/v) sodium chloride and subsequent precipi­
tation with 4 volumes of ethanol gave a white precipitate
which wae freed of protein as described earlier (see
page 18) and then hydrolysed with concentrated perchloric
acid#

The hydrolysate# when treated for the separation of

purine and pyrimidine bases (see page 30) gave a large
amount of end-absorbing material on elution with water and
a very slight peak at 260 mp after 20 ml. of 2 N hydrochloric
acid had passed through the column.

Since the bulk of the

72

®eberlal eppe&red to be polysaccharide in nature, an
alternate procedure was therefor© sought.
It was decided to start with th© intact mycelium and
to apply the method of deoxyribonucleic acid extraction
suggested by Charg&ff and Zamenhof (89) from bakers ye&st#
It was hoped that th© content of deoxyribonucleic acid in
Murospora would be greater than in yeasts, where the over­
all yield is only 0.18 per cent*

The mycelium, after

soaking in 0*1 M sodium citrate buffer of a pH of 7*5 to
inhibit deoxyribonuclease was ground with liquid nitrogen,
and the resulting powder was suspended in IS ml. of the
citrate buffer and centrifuged at 4,000 r.p.m. for 2 hours
* in a Sorrell refrigerated centrifuge^*

Th© procedure,

called differential centrifugation, was expected to give
three layer®, the middle layer consisting of fragmented
eelle and nuclei*

This situation was not realized, how­

ever, probably because of Insufficient cell breakage
during the grinding*

The total solids were therefore

centrifuged off at 10,000 r.p.m. and extracted with 1 M
sodium chloride for 72 hours in the refrigerator.

The

supernatant obtained from a second centrifugation at 4,000
r*p*m. for 2 hours did not give the characteristic poly­
meric threads of deoxyribonucleic acid on addition of
4 volumes of ethanol*

The apparent low concentration of

deoxyribonucleic acid in the mycelium prompted a decision

*Xvan Sorrell Inc., Hew York

73

to forego the investigation of more efficient grinding
methods*
The Effects of Belated Substances on
Growth of the Beurob p ora Mutant
In the course of the labeling studies a number of
possible metabolic relationships suggested themselves*
These were tested by growing the pyrimidine-requiring
mutant in basal media, as described previously (see page
26), and adding the compounds to be studied to the flasks*
Additions of heat-stable materials were made prior to
sterilisation by autoclaving, and heat-sensitive substances
were added asoptically after filtration through a fritted
glass bacteriological filter*

The cultures, generally run

in triplicate, were incubated at 25° C for 6 days*

The

mycelial mat of each flask was then removed, rinsed with
distilled water, squeezed to remove most of the water,
dried in a 55°C oven for 6 hours, and weighed*
Experiments were run in which alpha-hydroxybutyrie
acid was the only additive, in the event that alphaaroinobutyric acid might be utilized by way of the hydroxyacid*

Alpha-hydroxybutyric acid was synthesised from buty­

ric acid by the Hell-Volhard-Zelinsky procedure (60).
Twenty-five g. of butyric acid was placed in a 250 ml.
round-bottomed flask equipped with a reflux condenser.
Two grams of red phosphorus were then added followed by
gradual addition of the theoretical amount of bromine*

74

Th© flB.sk was cooled in an ic© bath until th© reaction sub*
sided, and the mixture was then placed on the steam bath
for the completion of the reaction*
process required 4 hours*

The entire bromination

The resulting alpha-broraobutyric

acid was slowly dropped with shaking into 100 ml* of hot

water in a 250 ml* flask over a 1-hour neriod,

The organic

layer was distilled at 10 mm* and the 78 to 84°C fraction
retained for hydrolysis*

Ten g* of the alpha-bromobutyrio

acid was treated with 8*5 g* of potassium carbonate in 50
ml# of water#

After 8 hours the mixture was acidified to

a pH of 2* evaporated almost to dryness* extracted 4 times
with ether* and the ether extract evaporated to small vol­
ume*

The oily liquid resulting was distilled in vacuo and

crystallized in the receiver m

long white needles * Be­

cause of the hygroscopic nature of the compound, a melt­
ing point was not obtainable*
Another experiment was prompted by th© studies of
Jones, Specter, and Lipmann (15) on carbamyl phosphate,
the synthesis of which was carried out as follows * Onetenth mole (13*6 g#) of potassium dihydrogen phosphate and
0*1 mole (8*1 g.) of potassium cy&nate were dissolved in
100 ml. of water and heated to 30°G for 30 minutes.

The

mixture was then cooled on ice and 0*2 moles of perchloric
acid containing 0,3 mole® of lithium hydroxide was added.
The white precipitate of potassium perchlorate and lithium
phosphate was removed by filtration, and th© solution of

76

lithium oarbamyl phosphate was precipitated by slow addi­
tion of an equal volume ot ethanol*

The crystals were

filtered off, redissolved, reprecipitated by ethanol, refiltered, and dried in a dessicator over calcium chloride*
The yield wae 7*1 g«, 48 per cent of the theoretical value*
The effect of this compound on the rate of growth of the
mutant on amlnobutyrie acid was studied in the event that
earbamyl phosphate might donate a carbamyl group to this
acid in the formation of the pyrimidine ring*

Further­

more, Fairley (26) had demonstrated the powerful inhibi­
tory effect of arginine on the growth of this mutant on
amlnobutyrie acid, and it was of interest to see if added
carbamyl phosphate overcame this inhibition*

The work of

Grisolia and Wallach (61) also implicated carbamyl phos­
phate as the donor of a carbamyl group to beta-alanine
to form beta-ureldopropionic acid, a pyrimidine precursor,
and the mutant was therefore grown with various combina­
tions of these substances*

The synthesis of beta-

ureidopropionic acid was carried out by the reaction of
potassium cyanate with beta-alanine, as described by
hengfeld and Siieglitz (61).

Two g« of beta-alanine and

1*05 g* of potassium cyanate were simply placed in an evap­
orating dish and evaporated on the steam bath to a syrup*
fills residue was transferred to a small beaker and placed
in the refrigerator*

The resulting crystals were separated,

rediesolved in hot water, filtered, and the solution again

76

placed in the regrigerator.

The yield of beta-ureidopro-

pionic acid wae 1*6 g*, a 62 per cent yield*

The melting

point was 169-170°C.
A series of vitamin®, including folic acid, pyridoxine,
thiamine, and vitamin B^g were also studied as to their
effect on the growth of the mutant on uracil, amlnobutyrie
acid, and uridine*

Folic acid and thiamine were of particu­

lar interest because of the possession of a pyrimidine struc­
ture in the molecule, and vitamin Big was Implicated by the
worts of Jukes et al. (62)*
The results of the growth studies are presented in
Table VII*

It can be seen that alpha-hydroxybutyric acid

does not replace the pyrimidine requirement of ffeurospora
1298*

Furthermore, ©arbamyl phosphate does not stimulate

growth on amlnobutyrie acid nor does it overcome the in­
hibitory effect of arginine*

The combination of beta-

alanine and oarbamyl phosphate was also Ineffective in
replacing the pyrimidine requirement*

The vitamin studies

suggest that thiamine and vitamin Big have no effect on
th© utilisation of amlnobutyrie acid*

However, folic acid

has a marked stimulatory effect on growth with both amlno­
butyrie acid and uracil, and not with uridine, though the
effect is noted only at low levels of either precursor.
Pyridoxin© has a surprising inhibitory activity in the
case of growth on amlnobutyrie acid.

TABLE VII
THE EFFECT OF VARIOUS COMPOUNDS OB GROWTH
OF IECBOSPORA 1898
Growth as mg.
I

Ion#
I Jag« hydroxybutyric acid

2

Ion#
& mg* amlnobutyrie a d d
n
"
" *8 fflg# earbamyl
phosphate
2 Dig* earbaayl phosphate

0
30

Ion#
I mg* amlnobutyrie a d d
»
tt
* - 5 jug arginine
n
«
it «
*
a
- 2 mg* oarbamyl phosphate
ii
amlnobutyrie acid - 5 Mg. arginine
• 20 mg* oarbamyl phosphate

0
23
0

Bone
3 mg* amlnobutyrie a d d
**
* * 2 mg* oarbamyl
phosphate
a beta-alanine
«
*
- 2 mg* oarbamyl phos­
phate
oarbamyl
phosphate
2 *
beta-ureidopropionie acid
3 *

0
32

3

4

0
0

31
0

0
0

54

0

0

0
0

Bon#
2
1 mg# amlnobutyrie acid
»
#
#
- 1 jag Vitamin Big
6
«
»
* - 2 m
9
* 1 2
a h
#
»
27
nil
»
» - ! w w
*
SI

78

$HE EFP&CT OF VARIOUS COMPOUNDS OH GROWTH
OF mVBQ&FQBA 1298 (COM1.)
Expt#
L* j|ui

Growtii as mg.

Hone
5 mg* amlnobutyrie acid
«
n

n

*

- 0*1 mg* folie acid
- .02 *
M
■

0*1 mg# folie acid
0.02
*
«
7

lone
5 mg* amlnobutyrie a d d
tt
n
ft m
tt m*
a
»
»
»
tt m
It
8 mg*
*
»
H
tt m
ft
ft

Jt
«

*
n
»
tt

uracil

a
ft
ft

0*2
n mg#
° uridine
a
«
tt
»
»

»
tt

0*1 m* » folio a d d
0*5 it
thiamine
»
0*5
pyridoxin©

0.1
folie acid
0.5 © thiamine
Wtt 0*5 tt pyridoxin©

0.1 n folio acid
m 0. ® © thiamine
we. 0*5 fi pyridoxin©
•© 0*1 a
ee 0.5 »
* 0.5 n

folio acid
thiamine
pyridoxin©

0
22
51
20
O
0
0
28
m
27
11
13
24
13
4
37
51
33
41
16
14
15
15

It should be noted that negative result© such as those
obtained with oarbamyl phosphate and ureidoprooionic acid
are difficult to interpret# since the possibility always
exists# especially with charged compounds, that a permeabil­
ity barrier Is responsible for th© non-utilization of th©
compound tested*

70

2E&& H M M z ation of Aminobutyri c Aoid for
Pyrimidine Biosynthesis in the Rat
Materials
One male albino pat weighing 200 g. wae injected
intraperitoneally with 1 ml, of a water solution contain**
ing 0,1 me* (0*1 mM) of 3~c£4 alpha-aminobutyr1c acid
which was obtained commercially*.
Isolation of Deoxyribonuolelc Aoid
*fh© method of isolation was the same as that used in
the earlier methionine study (see page 11} •
Isolation of Purine and Pyrimidine Bases
fh© hydrolysis of the deoxyribonucleic aoid was
carried out as before (see page 13) but th© resulting per­
chloric acid solution of the purine and pyrimidine bases
was treated by the method outlined on page 30 since this
had been found to be a more useful procedure.
Purification of the Purine and Pyrimidine Bases
fh© fractions from the column separations were banded
on 7-inch sheets of Whatman Ho, 1 filter paper and chromat­
ographed in isopropanol - water as outlined earlier (see
page 33)#

•California Foundation for Biochemical Research,
Loa Angelea, California

80

Concentration and HadioacUvity Ucaaurementa
She t u w

were treated exactly «. outlined prevlouely

(see page 36) for the determination of specific activity.
hCSUltS

Th© activities of th© purine and pyrimidine bases
are shown in Table vill.

It is 8©©a that alpha-amino-

butyric acid is not a precursor in th© rat under these
experimental conditions*

The relatively higher labeling

in the cytosine might suggest that th© routes of synthesis
for thymine and cytosine are different, but the order of
activity is so low that th© validity of any such inter­
pretation of the data is highly questionable*
?A8£B VIII

INCGBFQBAT1ON OF AMINOBUTYRIC ACXD-3-C14
INTO NAT UNA COMPONENTS
^pacific Activity
Compound Isolated

----------- )

.

Adenine

42

Thymine

24

Cytosine
Amlnobutyrie
acid supplied

180

1.75

X 106

DISCUSSION

DISCUSSION
I M Utilisation of Methionine for
Itolne Biosynthesis
The results of th© study of th© utilization of methyllabeled methionine for th© biosynthesis of the purines and
pyrimidines of deoxyribonucleic acid indicate that the
methionine methyl carbon Is Indeed a precursor of the
ureide carbons of the purines and of the methyl group of
thymine.

This conclusion is based on th© observation of

significant labeling of these compounds after methionine
administration*

It has been assumed that the distribution

of activity i® the same for methi onin© -methy1 incorpora­
tion as that found for formic aeid incorporation into uric
acid by Bonne, Buchanan, and Delluva (65) and into nucleic
acid purines and thymine by Totter, Volkin, and Garter (26).
Support for the validity of this assumption is supplied by
the experimental finding of extremely low amounts of iso­
tope in cytosine.

Th© labeling of thymine and the purines

must, therefore, have been accomplished by means of a
specific process, rather than by random labeling which
would have been accompanied by labeling of the cytosine
as well.

In addition, that fact that both methionine and

formic acid gave rise to thymine which on degradation gave
highly labeled iodoform substantiates the view that the
activity wae located in th© methyl carbon in each case*

82

9k* metabolle interrelatlonshlp of formic acid and methio­
nine has also boon demonstrated by the work of Berg (28),
who has shown the conversion of formic acid to methionine
In pigeon liver, and by ieinhouse and Friedmann (64), who,
by a fonaate-trapping procedure, have found that methyllabeled methionine rapidly gives rise to labeled formate
in the urine of rate#

These studies clarified earlier

work by du Vigneaud

a|.* (66, 66} wherein it was demon­

strated that formate and formaldehyde could be converted
to a variety of "labile* me thy1-group donors including
creatine, choline, and methionine#

The latter le there­

fore clearly involved in the pool of "active one-carbon
unite* described by Tarver (67)*
However, it should be noted from the dilution values
that formic acid was used for purine synthesis to about
tea times the extent to which the methyl group of meth­
ionine was used*

On the other hand, a comparison of in-

oorporatlon of the two precursor® into thymine indicates
only a two-fold greater utilisation of formic aoid*

It

must therefore be concluded that methionine was utilised
for thymine synthesis by some route not Involving free
formic acid*

Further evidence for an alternate pathway

is supplied by the work of Elwyn and Sprineon (27) where­
in it was demonstrated that the beta-carbon atom of serine
is converted readily to the methyl group of thymine by a
mechanism allowing the retention of both hydrogen atoms

85

originally bound to this carbon atom, an impossibility
If the beta-carbon atom had boon oxidised to formic add*
$he»e results suggest conversion of both serine and meth­
ionine to some active one-carbon unit

at the oxidation state

of formaldehyde which may be readily converted to the methyl
group of thymine * Berg {28), on the basis of the Increase
of formate incorporation into both methionine and serine
eaused by homocysteine, postulated an S-hydroxymethyl
derivative of this compound as the active intermediate*
However, a considerable amount of work points to a role
for a hydroxymethy1 derivative of tetrahydrofolle aoid*
By the single addition of tetrahydrofolle acid, Kislluk
and Sakami (68) have been able to restore the ability of
Dowax 1-treated dlalyxed pigeon liver extracts,to combine
labeled formaldehyde and glycine to form labeled serine*
Formic acid could replace formaldehyde in this reaction
by the addition of adenosine triphosphate, diphosphopyridine nucleotide, glucose-e-phosphate, magnesium ion and
tetrahydrofolle acid*

These results suggest that formal­

dehyde reacts readily with tetrahydrofolle acid to give a
hydroxyme thy1 derivative, presumably ff&-hydroxymethyltetrahydrofolic acid, the active one-carbon unit at this
oxidation state, but that formate must first react in the
presence of adenosine triphosphate to form a formyl deri­
vative of tetrahydrofolle acid which is then reduced by a
diphosphopyridine nucleotide smsyme system to the

84

hydroxymethyl derivative.

Haraill (89) hue supplied

further cevldence for th© Involvement of this Intermediate
slmee labeled formaldehyde was found to he at least as
good a precursor of thymine as is formate in tide rat#
Such a route would appear unlikely if th© sol© route of
incorporation of formaldehyde required preliminary oxide*
bleu to formic acid*
Rachel© W i

the finding* by Lowry# Brown# and

that one deuterium atom is lost on utilise*

bion of dldeut©ro*e14*labele d formaldehyde for synthesis
of the thymine methyl group in the rat w a© explained as an
isotope offeat#

the direct interaction- of formaldehyde,

and tetrahydrofolle aoid has In feet recently been demon*
strated by Rialluk (71) * the product has been found to
function as the active on®-carbon donor In the conversion
of glycine to serine# and appears# therefore, to be the
intermediate through which both serine and methionine are
utilised for the synthesis of the thymine methyl group#
It Is interesting to note that hydroxywethyl derive*
tivee of uraoll (79) cytosine (75) and oytidin© (74),
which may represent the products of reaction of these
substances with the tctrahydrofo!ie acid derivative# have
been isolated from natural sources*
m e other possible mechanism of Involvement of
methionine in thymine biosynthesis# involving direct trans­
fer of the methyl group# can not be a quantitatively Import­
ant route for thymine synthesis since female acid, formal­
dehyde and serine are all utilised to a greater extent than

85

is the methyl group of methionine • These compounds are,
therefore* not utilized by prior conversion to methionine.
However, the reservation must be made that it is difficult
to determine the extent to which each of these metabolites
is utilized under normal conditions#

The sizes of the

metabolic pools of the various precursors may differ
widely, in which case a comparison of the incorporation
of isotoplo carbon contained in these compounds is a
questionable measure of the relative value of these sub­
stances as precursors of the compound studied#
Keeping this reservation in mind, it may be concluded
that methionine is a minor source of carbon atoms 2 and
8 of the purines of deoxyribonucleic acid#

The data for

thymine# however, suggests that methionine may play a
significant role in the biosynthesis of the methyl group
of this pyrimidine#
The tit1lizatlon of Amlnobutyrie Acid for
Pyrimidine Biosynthesis in Neurospora
The study of the labeling of the purines and pyrimi­
dines of th© ribonucleic acid of Heurosgora c.rassa strain
1298 after growth on alpha-aminobutyric acid-S-C14 indi­
cates a specific utilization of this precursor for bio­
synthesis of the pyrimidine ring in this organism#

it

may be seen in Table V that after the four-day growth
‘ period the isolated pyrimidines, uracil and cytosine, had
specific activities in the region of 5000 c,p,m,/)iM.

86

whereas the purlnes, adenine and guanine, had specific
activities of 1000 c#p*nu/pM.

The same labeling pattern

wae alec found in the nucleotide® of th© acid-soluble
fraction, the pyrixaidin© nucleotide , uridine-5 1—phosphate,
being labeled to the extent of 4000 c.p.nu/pM and the
purine nucleotide, adenosine-51-phosphate, having a
specific activity of S00 e*p«flw/pM.

The values for the

purines are about what would be expected if the labeled
amlnobutyrie acid was being utilised as a carbon source
at the same relative rate as the other carbon source® of
the medium - sucrose and tartrate#

Thie low order of

activity was likewise found for aspartic acid, glutamic
acid, and threonine isolated from the soluble proteins of
this organism#
The high order of labeling in protein Isoleueine,
isolated from the wild strain after four days growth and
from the mutant after a six-day growth period, is what
would be expected since Abelson and Vogel (76) and
Melberg

al# (57)had demonstrated that amlnobutyrie

acid is a normal intermediate in isoleueine biosynthesis
ia Meurocoora# The mechanism of its utilization, as pro­
posed by the latter authors and also by Straesraan, Thomas,
and felnhouse (76), involves a ketone condensation between
alpha-ketobutyric aoid (obtained from the smino acid by
deamination) and some activated form of acctaldehyde
followed by a pinacol-type rearrangement and then amination
to give isoleueine#

87

Another result of this study Is ths corroboration of
the postulate of Adelberg et al* (57) that Isoleuclne and
valine aye formed by different routes In ffeurosoora. The
low labeling found In valine from the soluble proteins
Indicates that amlnobutyrlc acid Is not a valine precursor*
whereas, as mentioned previously* the high labeling of
isoleuclne established the Involvement of aminobutyrlo
acid in isoleuelne biosynthesis#
However* as may be seen in Table 7* the labeling of
pyrimidines and Isoleucine was only one-tenth the value
of the precursor* suninobutyric acid*

Bht this dilution of

the Isotope la the course of its utilization is explained
by the finding that the specific activity of aminobutyrlo
acid in the acid-soluble fraction is also greatly decreased*
the value being 2300 c#p#m#/jiM at the end of the six-day
growth period*

This specific activity compares favorably

with the labeling observed in both uracil and isoleucine
obtained in the same experiment#

The dilution of the iso­

tope is therefore explained by a corresponding dilution
of the precursor in the course of growth* apparently by
endogenous aminobutyrlo acid#
In regard to the mutation in Jl# crass a 1298 which
brings about its pyrimidine requirement* it may be noted
that Mitchell and Houlahan (3) have described a number of
"partial block" mutants of Meurospora. The organisms are
suggested as being deficient in a single reaction step*
perhaps through a decreased affinity of enzyme for substrate.

88

This barrier in the metabolic pathway is surmountable by
means of a large excess of the substrate or by incubation
at a lower temperature, the latter being explained on the
basis of the heat-sensitivity of the ensyme.

In the pres­

ent study it seems plausible that the large excess of aminobutyric acid at the start of the incubation period enables
sufficient accumulation of the precursor to bring about
reaction at the partially-blocked step*

Once growth be­

gins the organism can presumably synthesise its own
aminobutyrlo acid, and therefore the pool of this pre­
cursor is subsequently diluted, with a consequent dilu­
tion in the labeling of uracil and cytosine*

This phenome­

non of an organism having the requirement of a substance
for growth and yet the ability to synthesise the substance
in the course of growth has also been demonstrated by
Bonner, Yanofsky and Partridge (77) in tryptophaneless
mutants of Maurospora* The authors describe this occur­
rence as nleakage**, and Haldane (78) suggests that such
a phenomenon prove® the ovep-similicity of the "metabolic
block” idea*
This combination of a "partial block** of some reaction
step on the route to pyrimidines with the "leakage** phenome­
non as an explanation for the behavior of this Heurospora
mutant is not, however, without its uncertainties*

It has

been noted that the labeling of the aminobutyric acid re­
covered from the mycelium after six days growth was of the

89

same order of magnitude as the labeling of the pyrimidines.
It might be expected that the formation of pyrimidines
and of aminobutyrie acid would be processes occurring in
relatively constant proportions throughout the growth
period*

Under these conditions it would be expected that

the labeling of the pyrimidines would be higher thfln the
labeling of the aminobutyrie acid, since some of the
pyrimidines would be formed at earlier stages of growth,
when the aminobutyrie acid pool had not been diluted to
the final extent • The fact that this result is not ob­
tained suggests, therefore, the greater part of the
dilution occurred at a stage in growth before much
pyrimidine biosynthesis had occurred*
The suggestion might be made that aminobutyrlo acid
or some closely related derivative possesses an additional
role as a catalytic factor.

The greater-than-additive

effect of combined uracil and aminobutyrie acid on the
growth of the mutant, noted in earlier work (£4) suggests
a dual role for this amino acid.

In addition, the much

greater growth response of the mutant to uridine suggests
that the latter, or some closely related derivative, may
be the catalytic factor to which aminobutyrie acid is con­
verted.

The importance of uridine-containing eoenzymes

in metabolism has been suggested by the demonstration of a
whole series of naturally-occurring uridine diphosphate
derivatives where the latter Is combined with glucose (79),
galactose (80), aeetylglueosamine (81), and an amino sugar

90

which is in turn Joined to a series of amino acids (82)*
A group ©f compounds similar to the last have also been
demonstrated by Binkley (83) to result by the digestion of
hog kidney with a proteolytic enzyme*

The fact that those

substances also had enzymatic activity suggests that uri—
dine derivatives may form the core of some ensymss* and
that therefore the involvement of such substances in
enzyme synthesis may not be due solely to their function
as nucleie acid constituents#

A catalytic role of amino*

butyric acid may thus result by virtue of its initial
conversion to a uridine-containing compound which may
either act as a cofactor or become a part of an enzyme
involved either directly or Indirectly in aminobutyrie
acid synthesis#

This additional effect might therefore

explain the synergism noted in growth of the mutant on
combined uracil and aminobutyrie acid#

It also satisfies

the requirement of early dilution of the precursor, men­
tioned previously as the probable explanation of the
similar specific activities of aminobutyrie acid and
pyrimidines#

If aminobutyrie acid was also acting In

an autocatalytic manner to form more aminobutyrie acid,
early dilution of the precursor would certainly occur#
Granted that aminobutyrie acid i® a precursor of
pyrimidines iw JJeurosgora, it then becomes of Interest to
consider the possible reactions for conversion of the
amino acid to the pyrimidine ring#
The low labeling found for orotein aspartic acid in

91

all experiments precludes the possibility of conversion
of aminobutyrie sold to asp&rtio sold as the route of its
utilisation.

If this amino acid or a closely related

derivative were involved, the labeling of the aspartic
acid pool should be of the same order of magnitude as
the aminobutyrlo acid - shown to have a specific activity
of 2500 e*p.nu/uM*

the aspartic acid pool should also

be in equilibrium with the Neurogpora proteins, and the
aspartic acid of the latter should therefore also be
highly labeled*

Since this is not found to be the case,

a specific activity of 500 c.p.au/uM being found instead,
free aspartic acid is apparently not an intermediate in
the conversion of aminobutyrlo acid to pyrimidines*
However, the selective labeling of the pyrimidine
bases and nucleotide® is in agreement with the possibility
that the carbon chain of aminobutyrie acid is utilised
as & source of the pyrimidine ring*

If aminobutyrie acid

may be considered to be utilised In a manner similar to
that demonstrated for aspartic acid in

orotlcuza by

IdSberraan and Komberg (84), the labeled beta carbon atom
should appear as carbon 5, the middle carbon atom of the
three-carbon chain of the pyrimidines« However, degrada­
tion of the isolated uracil indicates that the label,
though concentrated in the carbon chain, Is distributed
about equally between two carbon atoms of the chain*

This

result does not appear reasonable, though Lagerkvlst et al.

92

(1$) have reported results which suggest a much greater
utilisation of the methylene carbon than the carboxyl
carbon of aspartic acid—2>~ci®—4—cii for pyrimidine bio­
synthesis in the rat*

It is possible that cleavage of the

carbon chain of either four-carbon precursor occurs to
give two two-carbon fragments, and that the part consisting
of carbon atoms 3 and 4 then combines with a like fragment
to give the doubly-labeled chain*

In the case of amino-

butyric acidp cleavage might be preceded by conversion to
threonine by means of dehydrogenation and subsequent hy­
dration of the resulting beta-gamma unsaturated aoid*
threonine might then give rise to acetaldehyde9 a reaction
demonstrated In leurosoora by fagner and Bergquiet (85).
However, the selective recombination of fragments to give
a doubly-labeled compound must be regarded with considera­
ble doubt*

On the contrary, the results of considerable

metabolic study point to an interrelationship between amino
aeld metabolism and pyrimidine biosynthesis such that amino­
butyrie acid would be expected to give rise to the 5-labeled
pyrimidine*

The degradation results mi#it thus be explained

on bases such as the presence of an impurity in the pyrimidine
or the unreliability of the degradation procedure.

The for­

mer possibility seems unlikely in view of the conformity of
the absorption ratios of the isolated compounds to those of
known compounds $ because of the close agreement of radio­
activity and concentration of the column ©luates, and by

93

Virtue of the constancy of specific activity after repeated
paper chromatography in several different solvent systems •
fhe results therefore seem to case some doubt on the re­
liability of the degradation procedure, at least when
carried out on this small scale.
the work of Mitchell and Houlahan (7) implicated
oxalacetic acid and aminofumari c acid as pyrimidine pre­
cursors in Heurosgora, and subsequent studies by Lagerkvist
•t el# Cl®) and by Woods, Ravel, and Shive (13) demonstrated
the involvement of aspartic acid in pyrimidine biosynthesis,
fhe mutant under investigation in the present work, H. orasstrain 1298, has been shown to utilize, in addition to
aminobutyrie acid, either threonine or homoserine but not
aspartic acid for growth (24, 28),

The central role of

homoserin© in the biosynthesis of methionine and threonine
was suggested by the work of fees, Horowitz, and Fling (86)
wherein homoserine was found to replace the joint threonlnemethionin© requirement of a leurospora mutant.

More recent­

ly Abelson and Vogel (75), by the use of an isotope-corapetition technique, have demonstrated a biosynthetic sequence
jp Meurospora consisting of aspartic acid, homoserin©,
threonine, alpha-ketobutyric acid, alpha-k©to-beta­
me thylvaleric acid, and isoleucine.
found to be converted to methionine*

Homoserine was also
In addition, the meta­

bolic formation of homoserine and aminobutyrie acid from
methionine has been demonstrated by Matsuo and Greenberg (87).

94

The relationship between aminobutyrie acid, homo*
serine and threonine explains the ability of Ji, orassa
1998 to utilise any of these three amino acids for
pyrimidine biosynthesis*

In addition, since the magni­

tude of the growth response was similar for all three
compounds, and sine© aminobutyrie acid was shown in the
present work to be a pyrimidine precursor, the possibility
exists that all three amino acids may be converted to a
common intermediate in the course of pyrimidine biosynthe­
sis • Teas jJ; al« (86) have suggested that threonine may
be converted to homoserine by dehydration to the beta-gamma
unsaturated acid and subsequent hydration of the double
bond*

A similar dehydration reaction has been suggested

by Chargaff and Sprlnson (88 ) as involved in the deamina­
tion of serine#

The utilisation of aminobutyrie acid

might proceed by dehydrogenation to the beta-gamma unsaturated acid followed by hydration to homoserine#
Furthermore, the reversible conversion of homoserine to
aspartic acid has recently been demonstrated in yeast
extracts by Black and Wright (89).

The primary alcohol

group of homoserine is oxidised to an aldehyde group,
giving aspartic acid beta-semtaldehyde. ,This compound is
then oxidized in the presence of inorganic phosnhate to
the corresponding acyl phosphate, beta asoartyl phosphate,
and the latter is subsequently dephosphorylated to give
aspartic acid.

The final step in this sequence of reac-

95

tien* can not, however, occur reversibly in Neurospora.
The fact that in the present study aspartic acid from the
soluble proteins is only randomly labeled while the amino­
butyrie aeid pool has a specific aotivity of 8300 e#p.ra./uM.
is not compatible with the idea of a rapid interconversion
of the two metabolites * The conclusion therefore seems
reasonable that the enzyme for one of the steps in the
reaction sequence from homoserine to aspartic acid is
not present in ieurospora.
The route of utilization of aminobutyrie acid for
pyrimidine biosynthesis In ffeurospora may therefore in­
volve homoserine and also one or more of the previouslymentioned aspartic acid derivatives*

A reactive compound

such as beta-aspartyl phosphate may readily react with
carbon dioxide and ammonia by cleavage of the phosphate
bond to give ureidosueclnio acid*

The latter might then

undergo cycllsation to dihydroorotic acid and then oxida­
tion to orotic acid, as demonstrated in Z. oroticum by
Ueberman and Komberg (84).
The low labeling found for threonine from the soluble
proteins suggests that aminobutyrie aeid is not readily
convertible to threonine under these experimental condi­
tions, but that hydration of the beta-gamraa unsaturated
intermediate may occur in such a manner as to give only
homoserine*

The possibility also exists that the unsatura—

ted intermediate may not undergo hydration to either

96

hemoserine or threonine in this organism, but that this
reactive compound is instead in some way directly utilised
for the formation of the pyrimidine ring*

However, in

lieu of the known role of homoserine as a methionine preeursor in ffeurospora. the tentative identification of
highly labeled methionine sulfone in the aeid-soluble
fraction of J|* crassa 1298 in the present work suggests
that conversion of aminobutyrie aeid to homoserine must
actually occur in this organism*
Another possible pathway whereby aminobutyrie acid
may be utilised for pyrimidine biosynthesis is suggested
by the studies of Fink jt al* (SO, 90} wherein it was
demons trated that dihydropyrimidines, be tenureido acids
and beta-amino acids result as reduction products of
thymine and uracil in rat liver*

fhese authors suggested

that this may represent not only a degradative pathway
for pyrimidines but also a method for their synthesis*
Uhls suggestion was strengthened by the reeent work of
arisolla and Wal3a oh {31} in which they demonstrate the
reversible conversion of dihydrouracil and beta-ureidopropionic acid in beef liver extracts*

Furthermore, erude

extracts were shown also to convert beta-ureidopropionic
aeid to beta-alanine, and these authors were also able to
demonstrate the reverse reaction in rat liver mitochon­
drial preparations and in bacterial extracts (91) upon the
addition of carbamyl phosphate*

It would therefore appear

97

that beta-alanine way be a precursor of the pyrimidine
ring*
Since beta-alanine has been shown to arise by the
deearboxyl&tion of aspartic acid in OX. welch 11 (92 ),
the mediation of the, previously-mentioned beta-as partyX
phosphate in aminobutyrie aeid utilisation seems quite
possible*

Aminobutyrlo aeid might be converted to

homoserine and then to beta-aspartyl phosphate, and the
latter might then undergo decarboxylation of the carboxyl
group representing carbon 1 to give a phosphorylated de­
rivative of beta-alanine.

Addition of carbon dioxide and

ammonia, perhaps aa carbamyl, phosphate. If necessary,
would yield beta-ureidopropionic acid, which could then
be eyelislaed and oxidised to f o m the pyrimidine ring.
M

the present study, the attempt to replace the pyrimidine

requirement of Jf. erassa 1293 with a mixture of carbamyl
phosphate and beta-alanine was unsuccessful.

Woods, Ravel,

and Shive (13) noted a similar inability on the part of
the aspartic aeid-reqnlriug L. arablnosus 17-5 to utilize
either beta-alanine or ureidosueoinie aoid.

However, these

negative results are of little significance since they quite
possibly may have been due to the inability of the compounds
to pass through the cell wall of the organism.

Such per­

meability barriers are known to be significant where highly
charged and polar substances are concerned.

98

The conclusion therefore seems reasonable that aminobutyric acid is utilised for pyrimidine biosynthesis by
conversion to homoserine followed by oxidation and perhaos
phosphorylation of the gamma carbon atom to give a reactlve Intermediate such as beta-aspartyl phosphate*

The

latter may then proceed to pyrimidines directly by reac*
tion with carbon dioxide and ammonia with subsequent
cyclisatlon and oxidation*

Alternatively, beta-aspartyl

phosphate might undergo decarboxylation to form a deriva*
tlve of beta-alanine which might then react with carbon
dioxide and ammonia to form beta-ureldoproplonlo acid
followed by cyclisatlon and oxidation to give the pyrimi­
dine ring*
In connection with this suggestion of a different
route for pyrimidine biosynthesis in Heurospora, it might
be noted that Davis (98) has observed that lysine bio­
synthesis in Neuroaoora involves an entirely different
pathway from that which exists in E* poll* He has demon­
strated that dlaminopimelic acid is an intermediate in
lysine biosynthesis in E* coll, whereas Good, Heilbronner
and Mitchell {94} have shown alpha-aminoadipic acid to be
involved in lysine biosynthesis in Heurospora* The same
difference has also been noted between £• utilus and N.
orassa by Abelson and Vogel (7b).

Furthermore, the obser­

vation by Woods, Havel, and Shive (15) that lysine - in
addition to threonine and pyrimidines - spares the aspartic

99

aoid requirement of L. arabinosua 17-5 suggested to those
Authors that lysine was involved in pyrimidine biosynthesis*
Mitchell and Houlahan (95) had earlier demonstrated the
aeoumulation of pyrimidines by lysineless mutants of
12HS2SJ22£&* and heermaim reported an Inhibitory effect
of arginine in Neurospora similar to that observed by
Fairley (85) with the pyrimidineless mutant used in the
present work*
The fact that a definite difference exists between
Heurospora and other organisms in regard to lysine bio­
synthesis , when coupled with the observed pyrimidinelyslne relationship, suggests that the unusual biosynthe­
tic pathway for both pyrimidines and lysine in ffeurospora
may be attributable to some alteration in an intermediate
common to both pathways*

The studies of Abels on et al* (96),

wherein lysine was shown by isotopie competition experiments
to be an aspartic acid metabolite, indicates that aspartic
acid or some derivative may be this common intermediate,
The involvement of such an intermediate in lysine biosyn­
thesis remains to be clearly demonstrated* but the lyelnepyrimidlne relationship at least serves to demonstrate the
existence of real differences in biosynthetic pathways in
different types of living things*
As may be seen in Table V, the labeling of the purines
and pyrimidines of the wild strain, while not as markedly

100

different as In the ease of the mutant, nevertheless
demonstrates that aminobutyrie acid is a normal precursor
of pyrimidines In this organism,

The finding of amino-

butyric aeid as a component of the free amino acid pool
of the wild strain * grown without aminobutyrie aeid also supports this suggestion.
However, regardless of the pathway of utilisation
of aminobutyrlo aeid for pyrimidine synthesis, it seems
reasonable to conclude that this anino acid must be a
significant precursor of these nucleic acid constituents
in Heurospora. The fact that the labeling of isoleucine for which aminobutyrie acid is a known precursor - is
similar in magnitude to that of the pyrimidines must be
regarded as strong support for a prominent position for
aminobutyrlo acid in pyrimidine biosynthesis in MeUrospora.
The Utilisation of Aminobutyrie Acid for
pyrimidine Biosynthesis in the Rat
The finding that aminobutyrlo acid is not readily
utilised for pyrimidine biosynthesis in the rat is not
unexpected since the previous study strongly suggests a
special situation for pyrimidine biosynthesis in Neurospora.
The pathway involving aspartic acid, demonstrated by
Lieberman and Romberg (17, 84) in Z. orotlcum, is undoubt­
edly also the major route for pyrimidine biosynthesis in

101

till# rat*

Weed end Wilson were able to demonstrate utiliza­

tion of orotic acid (7) and of ureidosueeinic acid (11)
for the synthesis of polynucleotide pyrimidines in rat

spleen, and Heichard (14) recently reported that rat liver
mitoohrondria are capable of converting aspartic acid,
carbon dioxide, and ammonia to ureidosuecinio acid*
The dilution of isotople aminobutyrie aeid on con­
version to rat deoxyribonucleic aeid cytosine in the
present study is approximately ten times as great a a that
found in earlier studies of methionine utilisation for
thymine biosynthesis.

This represents a dilution by about

5000 times In the course of utilisation, whereas methionine
was diluted over 25,000 times in the course of conversion
of the labeled methyl group to the ring carbons of cytosine.
There thus appears to be some slight selectivity in the me
of aminobutyrie acid in the rat, particularly since cyto­
sine was labeled to four times the extent of adenine.

The

lower specific activity of isolated thymine suggests a
different route of pyrimidine biosynthesis for the two
major deoxyribonucleic pyrimidines, thymine and cytosine.
However, the specific activities of the isolated com­
ponents are so low that the above interpretation must be
regarded as extremely tentative*

A more definite conclu­

sion must await further study of an inobutyric acid and
pyrimidine biosynthesis in the rat.

SUMMABY

SUMMABY
Methionine-mefchyl-C14 has been shown to give rise to
bignifleant labeling of the purines and thymine of
deoxyribonucl©1c aeid in the rat*

The data suggests

that utilisation of the methionine methyl carbon
occurs by way of the wl-carbon pool", but that formic
aeid is not an intermediate in toils process*

Rather,

an intermediate at the oxidation state of formaldehyde
appears more likely*

By comparison with formic acid,

the methionine methyl group appears to be of negligible
importance in purine biosynthesis, but the data for the
pyrimidines suggests that the methyl group of methio­
nine serves as a significant precursor of the thymine
methyl carbon*
A pyrimidineless mutant of Reuros pora crass a has been
shown to utilize alpha-aminobutyric acid-3-C^ for
pyrimidine biosynthesis*

The data suggest® that free

aspartic acid is not an intermediate in this conversion,
but an aspartic acid derivative, derived from aminobutyric acid by way of homoserine, appears as a reason­
able choice for one of the intermediates involved*

The

importance of aminobutyrie acid as a pyrimidine precur­
sor in heurosoora is indicated by the similar magnitude
of the labeling of isoleucine and the pyrimidines.

The

103

biochemical nature of the mutation possessed by N.
erasb a 1298 and possible reasons for the ten-fold
dilution noted on conversion of the precursor to the
pyrimidine are discussed*
3.

Alpha-aminobutyrie acid-3-C^4 was also studied as a
possible pyrimidine precursor in the rat*

Some

selectivity In utilisation of the isotope for cyto­
sine synthesis was found, though the specific activi­
ties of the compounds isolated were of such a low
order of magnitude that any interpretation of the
data must be qualified as highly tentative*

BXBLXO&fUtm

104

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APPSSDIX

APPENDIX
A*

The molar extinction coefficient® for thymine and the

purine* in the eluting solvents were determined as follows.
In the ease of thymine a IB mg. sample of the reerystallized base*was dissolved in 26 ml* of 0*1 Sf hydrochloric
acid*

One 5 ml* aliquot was diluted to 100 ml* with 0*1 N

hydrochloric acid and the optical density determined at
260 mp*

A second 5 ml* aliquot was diluted to 100 ml* in

0*015 M ammonium formate buffer of pE 8*25* and the optical
density at 260 mp. also determined*

The molar extinction

coefficient was then calculable by the following relation­
ship*
**•*»«£&.

°a
where ku * molar extinction coefficient in eluting
solvent buffer of pH 8*25
k

• molar extinction coefficient in 0*1 H
hydrochloric acid

©

z optical density at 260 mjx in 0*1 B
hydrochloric acid
% optical density at 260 myt in eluting
solvent

The procedure for adenine was similar except that

^Nutritional Biochemical® Corp.* Cleveland* Ohio

Ill

10 mg* of adenine sulphates was dissolved in

ml* of

0*X H hydrochloric aoid and dilutions war# made to 100
ml* with 0*1 N hydrochloric acid and 4 IT hydrochloric
acid*

The optical densities were again determined at

260 si^u

For guanine* 10 mg* of

guanine hydrochloride4*"*

was dissolved in 26 ml* of 0*1 H hydrochloric acid* and
dilution was made with 0*1 H and 6 N hydrochloric acid.
In this ease the optical densities were measured at 249 mji*
the wavelength of maximum absorption for guanine in acid
solution*
B*

The foraula used to convert the observed counts of

the purines and pyrimidines to specific activity.
_

25 «

Go x k x 1000 ml*

........

0

x

106

where S « specific activity (counts/minut e/micromole)
Co « observed counts {counts/minute/al«of sample)
k m molar extinction coefficient
D « optical density of sample counted
10® » factor to convert moles to micromoles
Sample calculation:
Co » 104 e*p*nu/ml* of sample
D • 0*693
104 x 7 X 103 X 1000
S •
... .
0.600 * 10®

thymine

k « 7 x IQ5 (SSCte^pH 8*25)
, ..
• 1050 c.p.m./uM

*BIoa Laboratories* Hew York
*HiEastman Kodak Co., Rochester, Hew York