——
——
——
———-—-—
——
——
——
——
——
——
——
——
——
——
——
——

ARGENENE AND mmmm mommasns
m NEURQSPORA AcmssA 1m

-1_.\__\
I40
1cn—Ao

 

 

Thai: {0? the Daqvee 0‘ M. 5.
MICHIGAN SUITE UHWEBSETY

A. Birk Adams

1959

 

 

  
   

LIBRARY

Michigan Stats
University

 

 

ARGININB AND PYRIMIDINE BIOSYNTHESIS IN

NEUROSPORA CRASSA 1298

By
A. Birk Adams

A THESIS

Submitted to the College of Science and.Arts of Michigan State
University of Agriculture and.Applied Science in
partial fulfillment of the requirements

for the degree of
MASTER OF SCIENCE
Department of Chemistny

1959

ACKNOWLEDGMENT

The author wishes to express his sincere appreciation to Dr.
James L. Fairley for encouragement and stimulating guidance through-
out the course of this work.

The author also wishes to express his appreciation to the var-
ious other members of the Chemistry Department for assistance and

helpful suggestions.

VITA

The writer was born in.Steubenville, Ohio, January 16, l93h and
received his secondary education at.North Canton High School, North
Canton, Ohio. He entered Bethany College, Bethany, West Virginia in
1951 and received his 3.5. degree in 1955 from this institution.

In the fall of 1955 the author entered the Graduate School of
Michigan.State University. After one year of study and serving as
a Graduate Teaching.Aseistant he was inducted into the United States
Army for a period of two years during which time he served as a clerk-
typist and medical technician in Stuttgart, Germany. He then returned
to Michigan State University in the summer of 1958, again being employed
as a Graduate Teaching Assistant in Chemistry while pursuing his stud-

ies.

ARGININE AND PYRIMIDINE BIOSYNTHESIS IN
NBIROSPORA CRASSA 1298

BY
A. Birk Adams

AN ABSTRACT

Submitted to the College of Science and.Arts of Michigan State
University of Agriculture and Applied Science in
partial fulfillment of the requirements
for the degree of
MASTER OF'SCIENCE
Department of Chemistry

1959

an... 791w a $4444

 

ABSTRACT

The purpose of this study was to gain information concerning the
mechanisms by which the mutant organism, Neurospora M 1298, forms
pyrimidine compmmds from the precursor, propionic acid. L-arginine had
been known to inhibit the growth of this organism in basal medium sup-
plemented with propionete, presumbly by blocking a reaction necessary
in the conversion or propiomte to pyrimidines. Evidence has been ob-
tained in the present work which supports this suggestion and eliminates
certain other possibilities.

Use was ads of this finding in attempts to identify intermediates
of the reaction sequence leading from propionate to pyrimidines. The
mold was supplied with propionate and argininc and a search was node,
using paper chromatographic techniques, for the acmlation in the
growth medium and in the nyccliul oi' the mold of compounds of the syn-
thetic process occurring in the sequence prior to theblocked motion.
No quantitative or qualitative differences were found, in a number of
emeriments, between compounds formed by the inhibited mold and those
formed by the uninhibited control until use was made of radioactive
propionic acid. 'ihe mold was supplied with propionatc--2—Cm and the
radioactive compounds formed from this by'the notabolic activities of
the mold and liberated into the medium were separated by two-dimensional
paper chrometograplw and located by radioautograpiw.

A new radioactive compound was detected on the chromatograms of
the arginine-inhibited mold, a conpound not present in the control.
Presumbly this compound is an intermediate in prOpionate utilization.
It has not, as yet, been identified.

TABLE OF CONTENTS

. Page
In mom-[ION I I I I I I I I I I I I I I I I I I I I I I I I I 1

WWWNTAL AND RESULTS a e a e e e e s s e e . e . s e a . o
GrWth Studie. on the M91111!“ “feet 9 e s e e e s s w s
Organism. e e e e e e a a e a e e e e e e e e a e e e

Materials...o..................

oo-q-qq-q

GTO"th.PrOCOdUr¢8 e e e s s s e e e a e e s e s e s 0

Experiments on Arginine Inhibition of Propionate
UtiliZItion s e e s e e e e e e e e a e a e e s e 9

Possible Alterationof the Mutant . . . . . . . . . . 13

Experiments on the Arginine Effect with Unlabeled
Prqpionate a e e e e e e e s e e s a e e e e e a s a s a 15

HaterIBIIOeeseaeeeseeeeeesasses 15
Chromatographic procedures and Results . . . . . . . 15

Experiments on the Arginine Effect with Sodium
PrOPiomu‘z'cl4seeeeeeeeeseaeseeses 21

Materials......................21
PaperChromatograpiw................ 21
2h
DISCUSSION.......................... 26

Radioautograprv . . .

Smy I I I I I I I I I I I I I I I I I I I I I I I I I I I I 33
mum I I I I I I I I I I I I I I I I I I I I I I I I I 31‘

LIST OF FIGURES

Figure Page
1. Liebeman-Kornberg Pathway of Pyrinidine Bioevnthesis . . 2

2. Proposed Fatima? for Pyrimidine Biosynthesis in
21.6133831293.s....o.....o....... 3

3. Optimum Concentration Curve for Sodium Propionate . . . . 9

LIST OF TABLES

Table Page

I. Arginine Inhibition of g. crassa 1298, Growth
InitiatedbySpores......o........... 10

II. Arginine Inhibition of N. crassa 1298, Growth
Initiated by chlial‘Fra—flmen s . . . . . . . . . . . . 11

III. L—arginine Inhibition of Intact Hycelia . . . . . . . . . 12

IV. Arginine Inhibition of g. crassa 1298 at Different
Suge‘ or m I I I I I I I I I I I I I I I I I I I 13

v. Effect of Arginine on "Altered“ pg. crassa 1298 . . . . . 11.
VI. Identification of a Nimydrin-Positive Compound . . . . . 20

VII. Activity of Some Ninlwdrin-Positive Canponents of
a. crassa 1298 Incubated with Sodium Propionate--2--Cm . 23

INTRODUCTION

In recent years biochemical mutants have been used extensively
both for the stuchr of genetics and for the study of biosynthetic path-
ways of vital cellular components. One of the most useful types of
these mutants has been the one produced by Beadle and Tatum (l, 2, 3)
in 1910 by the X—radiation of the mold Neurospora m. Ihe basic
concept behind the use of these mutants is that all biochemical
processes are genetically controlled. Only those reactions can occur
for which the necessary enzymes are present, and the ability to produce
each of the enzymes is a separate inherited characteristic. Destruc-
tion of a single genetic unit, a gene, results in the failure to pro-
duce a certain enzyme and, in turn, in a block in a particular sequence
of biochemical reactions. The organism, however, is capable of essen-
tially normal growth if it is supplied with compounds which occur after
the blocked step in this sequence of reactions. Under these conditions
the intermediate compounds prior to the blocked reaction will often
increase in concentration until one or more is detectable by chemical
means.

The generally accepted pathway for pyrimidine biosynmesis in most
organisms is the scheme shown in Figure 1 (h). This pathway was pro-
posed by Lieberman and Kornberg and is supported by evidence from sev-
eral different workers using a variety of different organisms (5-13).

Considerable evidence has been accumulating, however, indicating
that the mutant organism, Neurospora m 1298, can synthesize

pyrimidines by a different, as yet unknown, pathway. This mutant of

 

 

 

 

 

HOOC—CHz HOOC-CHz HOOC-CHZ
'_ HHS \_ ' Carbamyl
o-c coon , Hzm-cn-coon Phosphate > HZN (gm-coon
Qxaloacetic acid Aspartic acid O~C~NH
Ureidosuccinic
Acid
l ~HzO
0 O ' O
E-cu 5-01 "
, , Ribose-S-phosphate / ,, 1m“ Ff“!
HN C-COOH 1 ~ -
\ . yI—pyrophosphtatte Hli (o: OOOH < \ mi CH COOH
O-C-h 0-C-NH 0-C~fiH
Ribose—S'-Phosphate Orotic Acid Dihydroorotic
Orotidine-S'-phosphate AC1d
0 l O NH;
n s l
as“ F“ PC“
ATP I NH '
HN CH ‘~ HN CH '——-—5-—%>
\ o ’7 \ o ATP HR~ EH
0-C-N 0-C-N O-C-N
t i u
Ribose-S'—Ph03phate Ribose-S'-(Phosphate)3 Ribose-S'-(PhoSphate)3
Uridine—S'-phosphate Uridine-Si-triphosphate Cytidine-5'-triphosphate

NUCLEIC ACID PYRIMIDINES

Figure l. Lieberman—Kornberg pathway of pyrimidine biosynthesis.

a common.bread mold.was originally characterized as requiring uracil (3)
in the basal medium for growth. It was studicdihrther by Loring and
Pierce (lb) and was found to grow at a much higher rate on uridine or
cytidine than it did on uracil. It was also found that orotic acid
would support growth only as well as uracil.

Fairley and co-workers (15, 16, 17) using the same organism have

shown that cL-aminobutyric acid, propionic acid and related compounds

are incorporated into the nucleic acid pyrimidines to a much greater
extent than into the purines or the amino acids of the proteins. As a
. result of these studies it was evident that this mutant did not synthe-
size pyrimidines by the Liebermaanornberg pathway and a new pathway

was proposed as outlined in Figure 2 (l7).

 

Propionic acid ‘ dc-aminobutyric acid
Propionyl-CoA. Homoserine
Acrylyl—CoA ;< /?-hydroxypropionic acid
\A
[3-alanyl-C0A

I

fi-alanyl-COA. ribotide

i
/Q-ureidoprqpiony1-CoA ribotide

i

Dihydrouracil ribotide

1

Uridine-S'-ph03phate

I

Nucleic acid.pyrimidines

Figure 2. Proposed pathway for pyrimidine biosynthesis in Neurospora
crassa 1298.

h

This scheme is closely related to the degradative path for uracil
found in other organisms. When uracil was incubated with rat liver
slices, [g-alanine was formed (18). If the labeled compounds were in-
cubated with rat liver preparations an interconversion of dihydrouracil
and fl-ureidopropionate was denonstrated, but the formation of fl-alanine
from.ureidcpropionate appeared to be an irreversible process (19). Al—
though these studies seem.to involve a purely degradative pathway from
uracil to IQ-alanine, it remains a possibility that the ribotides of
the compounds in this sequence could be utilized for the synthesis of
pyrimidine nucleotides. Indeed, Mbkrasch and.Grisolia (20) have re-
ported the incorporation of dihydrouracil ribotide and fl9aureidepr0pionic
acid ribotide into ribonucleic acid.by rat liver preparations.

Fairley (21) made the interesting observation that very small
amounts of L-arginine in medium supplemented with oL-amin0butyric acid
would completely inhibit the growth of y, SEEEEE'1298: but had less
effect when the mold was grown on medium supplemented with uridine.
Upon further investigation (18) of this phenomenon it was found that
when the mutant was grown on labeled uracil in the presence of arginine,
the specific activity of the cytosine isolated from the mycelium.was
the same as the specific activity of the added.uracil. However, when
grown in the absence of arginine the specific activity of the qytosine
was only about 75070 that of the original uracil. This indicated that
once growth had started, this organism.was capable of synthesizing
pyrimidines by an adaptive route from.simple compounds and.that ar-
ginine was able to block this route. It seems quite reasonable to

assume from the similarity of the effects of arginine that this

5
adaptive route is closely related to the synthetic route from propionate
and aminobutyrate.

It was also found that the specific activity of the amindbutyric
' acid isolated from the acid-soluble fraction of the nycelium when the
mutant was grown on labeled.aminobutyric acid was only about 5 “7° that
of the compound supplied. The nucleic acid pyrimidines isolated had
similar, low specific activities (16). Thus it appeared that aminobuty-
ric acid.was needed to stimulate adaptation and once this adaptation
had occurred the organism.was able to aynthesize all of its necessary
components from the basal medium by the same route used with amino-
butyrate.

The present study was undertaken with the aim of gaining further
information about the pathway of utilization of propionate and amino-
butyrate for pyrimidine synthesis by'E, 253332 1298. The approach to
this problem was to use L-arginine under different conditions to block,
in specific fashion, the sequence of reactions leading from the simple
compounds to pyrimidines with the view that intermediates prior to the
blocked reaction would accumulate to the point where they could.be de-
tected, isolated and identified. This technique has been used success-
fully, for example, in identifying an intermediate in purine biosynthe-
sis in.Escherichia 2211 inhibited.by a sulfa drug (22).

Closely related to this basic problem of the. mechanism of pyrim-
idine formation is the precise nature of the inhibitory action of
arginine, and therefore, the arginine effect has also been.a subject of
the present study. Arginine would.be of value to this work only if
its inhibitory effect was the result of the blocking of a specific re-

action along the biosynthetic route of pyrimidine formation.

6

It was therefore considered necessary to investigate the possibil-
ity that the action of arginine was to affect cell permeability, pre-
venting the absorption of aminObutyrate and propionate, and the chance

that arginine simply prevented the adaptive formation of a necessary

enzyme in the germinating spores.

EXPERIMENTAL AND RESULTS
Growth Studies on the Arginine Effect
Organism

Neurospora‘ggggga 1293 was the organism.used throughout this
study and is one of the many mutants produced by Beadle and Tatum in
l9h0 by X-ray treatment of the wild strain. It differs from the wild
strain in that it is unable to grow on modified Fries basal medium (23)
containing inorganic salts, sucrose and biotin unless the medium is
supplemented with a source of pyrimidines. The organism was maintained
on agar slants consisting of 2 073 agar and 0.1 ‘75 uracil in the basal
medium. It was observed that spores obtained from.uracil culture slants
required one or two days longer to begin growth in medium supplemented
with sodium propionate as compared with spores obtained from culture
slants containing either sodium propionate or oL-aminobutyric acid.
Because of this, culture slants containing 1 ‘7; DL-ci~aminobutyric
acid in place of the uracil were also maintained. The mold was trans—

ferred to fresh slants about every two weeks.
materials

The uracil used.was a product of the Nutritional Biochemical Corp—
oration and the DL-<L-amino-n-butyric acid was a product of the Eastman
Organic Chemical Company. L-arginine hydrochloride was from the Pfan-
stiehl Chemical Company. The sodium propionate was prepared.by neutral-
ization of lGN. sodium hydroxide with redistilled propionic acid and

recrystallization from a water-ethanol solution.

Growth Procedures

In performing growth studies the mold was grown in 125 ml. Erlen-
meyer flasks fitted with cotton plugs. Any heat-stable reagents being
used in the experiment and 25 ml. of basal medium were placed in the
flasks before sterilization. The flasks were then stopperedwith the
cotton plugs and sterilized by heating in an autoclave for twenty min-
utes or more at 12-15 pounds pressure.

The sodium propionate and other similar compounds which were un-
stable or volatile in the slightly acidic medium were added to previously
sterilized flasks after being filtered through a sterile, sintered-
glass, bacterial filter using standard aseptic techniques. Finally the
flasks were inoculated with 1 ml. of a suspension of spores of _N_. m
1293 in sterile water. Growth was allowed to proceed until a moderately
heavy nycelial pad had developed, usually four to six days, and then
harvested by filtering the contents of the flasks separately with suc-
tion through a sintered glass filter. The mycelia were washed several
times with distilled water and then rolled into pellets and dried on
a spot plate for a day at 60° C. The dried mycelia were weighed on a
torsion balance.

Since higher concentrations of propionate were known to inhibit
growth of the mold, it was desirable to learn the concentration of
propionate needed to give optimum growth so that this amount could be
used throughout the arginine inhibition studies. Six sets of three
flasks, each containing 25 ml. of basal medium and concentrations of
sodium propionate varying from 2.5 mg. to 12.5 mg. per 25 m1. of medium,

were inoculated with 1 ml. of a suspension of spores obtained from a

9
propionate slant. Growth was allowed to proceed for a period of five

days. The mycelia~ were harvested, dried and weighed and the average
weights of the triplicate samples were plotted against the concentra-

tion of sodium propionate as shown in Figure 3.

Figure 3

Mg . of
mycelium

28

16
12

 

 

o 2 11 6 8 151211;

Sodium propionate concentration in mg. per 25 ml. of medium.

Experiments on Arginine Inhibition of
Propionate Utilisation

The amount of arginine needed to give one-half inhibition was
determined by a growth study. In later experiments aimed at finding an
intermediate in pyrimidine biosynthesis this amount of arginine could
be used so that growth would continue, but at a highly inhibited rate,
allowing intermediates prior to the partially blocked reaction to con-

tinually increase in concentration until they were able to be detected.

10
Eight sets of flasks in triplicate were set up containing 25 ml. of

basal medium and varying amounts of arginine. After the flasks were

autoclaved, 1 ml. of a sterile solution of sodium.propionate was added
to each flask and the mycelium harvested after five days. The results
are given in Table I and shows that 0.001 mg. to 0.0005 mg. of arginine

hydrochloride in 25 ml. of medium is sufficient to give one—half inhibi-

 

 

 

tion.
Table I
ARGININE INHIBITIGN OF'N. CRASSA 1298,
GROWTH mxmrsn E? m
Arg.HCl/25Aml. medium 25y weight of mycelium
Mg. Mg.
1 ' o
0.1 0
0.005 1.7
0.001 h.5
0.0005 5.5
0.0001 10.5
0.00001 10.5
0.0 10.5
Control (0.0005 mg. Arg. only) 0

 

In order to eliminate the inhibition of spore germination as a
possible effect of arginine on growth of the mutant another experiment
was run using a mycelial suspension.as the inoculant. One mycelial
pad of the mutant grown for six days on.propionate medium was placed
asceptically into a small, sterile, Waring Blendor containing 50 m1. of
basal medium.and homogenized for to seconds. One milliliter of this
suspension was used to inoculate each of five sets of four flasks con-

taining different concentrations of arginine in media containing both

11
sodium propionate and aminobutyrate. The mycelia were harvested and
weighed after six days.

For comparative purposes, three sets of flasks containing sodium
propionate in the basal medium were inoculated with 1 m1. of a spore
suspension. The results presented in Table II show that mycelial frag-
ments were as effective as spores for initiating growth on medium sup-
plemented with propionate and aminobutyrate and that arginine has
essentially the same effect when inoculation is by mycelial fragments

as when spores are used for inoculation.

Table II
WIRINE INHIBITIw 0F NEUROSPCRA CRASSA 1298

 

 

 

Substrate Inoculant Arginine-HCl Height of wcelium
mg. 3190
A. Sodium Mycelial
Propionate fragments O 26
' l 0
10 0-
B. Amino Mycelial
butyric acid fragments 0 11.0
1 0
10 0
A. Sodium Spores
Propionate O 26
1 0
10 0

 

Because it was not clear if growth in the previous experiment was
actually initiated by the mycelial fragments or by the possible presence
of spores in the suspension, further evidence was needed that arginine

was not merely an inhibitor of germination. For this purpose experiments

12
on the effect of arginine on the growth of intact mycelia were performed.
Eight sets of four flasks containing basal medium supplemented with
sodium propionate were inoculated with a spore suspension and allowed
to grow for four days, at which time a moderately heavy mycelium had
developed. varying amounts of arginine were added and growth was al—
lowed to continue for two days more before harvesting. The results are
given in Table III. This table shows that arginine inhibited growth of
the intact mycelium, but a much greater amount was needed for this ef-

fect than.was needed to inhibit growth from spores or mycelial fragments.

Table III
ARGININB INHIBITION OF'INTACT.M%CELIUH

 

 

 

m.HCl[25 ml. medium E weight of chlium
mg. mg.
0 1.2.3
1 35.8
0.01 h2.9 .
0.0001 h1.7
Control (Wt. of mycelium at start) 25.7

 

In another experiment the effect of arginine at various stages of
growth was studied. This time eight sets of feur flasks containing
basal medium supplemented with.propionate were inoculated with spores.
One milligram of L-arginine hydrochloride was added to a different set
each day after inoculation. The mycelia from all flasks were harvested
at the end of seven days.

An.experiment similar to the one just described was run on medium
supplemented with 10 mg. of DL-<L~aminobutyric acid. In this case 0.01

lag. of arginine hydrochloride was added and the mycelia harvested after

13
four days. The results of these two experiments are presented in Table
IV. Here we see that arginine inhibited growth at all stages on medium
supplemented with.pr0pionate and apparently also on medium supplemented

with aminobutyrate, although the evidence is not as clear.

Table IV

ARGININE INHIBITION OF E, CRASSA.1298 AT
DIFFERENT STMIES 0F GROWTH

 

 

 

 

Substrate Time of addition Dry weight
1 mg. Arginine.HCl of mycelium
A. Sodium days after inoculation mg.
Propionate l 0
2 0
3 Trace
h 0.5
5 1.1
S 1/2 u.8
6 8.5
Control (No arginine added) 20.8
B. d“ “8.311110"
butyric acid 0 0
l l
3 32.1
h 38
Control (No arginine added) 38

 

Possible Alteration of the Mutant

From time to time during these growth studies, the unsupplemented
medium would show heavy growth. This was assumed to be the result of
contamination of the cutlure slant used, perhaps hy the.wildrtype strain,
and so the experiment was discarded as being invalid. Later however,
the thought occurred that the growth might have been the result of a1-

teration of the mutant allowing it to synthesize pyrimidines from

1h
simple constituents in a more efficient manner. The result of one ex-
periment in which growth appeared on the blanks is given in Table V.
It is seen that sodium propionate slightly inhibited growth of this
organism and that arginine plus sodium propionate completely inhibited

the mold when present at the start of growth.

Table V
EFFECT OF ARGININE 0N "ALTERED" E. CRASSA 1298

 

 

 

 

Supplement Time of arg. addition Dry weight*
of mycelium

days after inoculation mg.

6 mg. Na prop. l 0
6 mg. Na prop. 2 Trace
6 mg. Na prop. 3 - 14.3
6 mg. Na prop. h 7.2
6 mg. Na prop. h.5 32.1
6 mg. Na prop. 5 35.0
6 mg. Na prop. No arginine added 37.6
None No arginine added 1.7.1;

 

flThe mold was harvested after six days total growth.

To see if the wild strain would behave in a similar fashion,
flasks containing sodium propionate and arginine, separately and in
combination, were inoculated with spores of the wild-type strain.
Sodium propiomte had no effect at all at the concentration used
(6 mg./25 ml.) and arginine at a 1 mg. per 25 m1. concentration had a
slightly stimulatory rather than inhibitory effect.

15

Experiments On The Arginine Effect
With Unlabeled Propionate

 

Materials

The compounds used were the same as those described for the growth
studies. The organism was again mutant 1298 of Neurospora crassa. The
solvents and color reagents used for chromatography were those described

by Block, 33 El. (21;) in a laboratory manual of paper chromatography.
Chromatographic Procedures and Results

In various experiments thirty or more flasks containing basal
medium supplemented with sodium propionate were inoculated with 1 ml.
of a suspension of spores of the mutant and allowed to grow until
mycelia developed. At this time each of twelve or more mycelia of ap-
proximately equal size was lifted from its flask, rinsed in three suc-
cessive beakers of sterile, distilled water and finally placed in sterile
Petri dishes containing 10 ml. of an aqueous propionate solution and 1
mg. of arginine hydrochloride. As a control, twelve or more additional
mycelia of equivalent size were transferred in the same manner into
Petri dishes containing propionate only. Aseptic techniques were follow-
ed during the complete transfer procedures.

The mycelia were allowed to remin in the aqueous solutions at
room temperature for periods of from five to ten hours. At the end of
the incubation period the contents of the dishes containing propionate
and arginine were filtered collectively by suction through a sintered
filter, rinsed with water and the mycelial pad placed in acetone for

30 minutes and then dried in a desiccator.

16

To remove the propionate the filtered solution was first acidified
and then extracted once with one-half its volume of ethyl ether and
twice more with one-quarter quantities. The extracts were combined.

The aqueous solutions and the ether extracts were both evaporated nearly
to dryness on a flash evaporator and taken up in a small amount of water.

The mycelia were allowed to dry for at least a day and then ground
to a fine powder in a mortar with.120 mesh carborundum. The powder
was placed in a 12 ml. centrifuge tube and 5 m1. of ice-cold, 10 ‘76
trichloroacetic acid was added. The solution was kept cold in a re-
frigerator for thirty minutes with occasional stirring. The tube was
then centrifuged.and the supernatant solution collected. The residue
was extracted twice more with 5 ml. of the trichioroacetic acid solu-
tion for periods of five minutes each. The extracts were combined and
extracted three times with 5 ml. of ether to remove the acid.and then
were evaporated to dryness and taken up in 3 ml. of water.

The mycelia and the solutions of the controls, for which the in-
cubation solutions originally contained only propionate, were treated
simultaneously in the same manner as the arginine-inhibited samples
described earlier.

Paper chromatOQraphs of the aqueous incubation solutions, the
ether extracts of these solutions and the trichloroacetic acid extracts
of the mycelia were prepared for the arginine-inhibited samples and
the controls to determine if any differences existed between them which
might be the result of an increase in the concentration of an inter-
mediate compound in the biosynthetic route of'pyrimidine formation. One-
dimensional chromatograms were first prepared fer screening the differ—

ent solvents and color reagents to be used.

17

The prepared solutions were applied to 18 inch long strips of
Whatman.No. 1 filter paper in dropwise fashion, drying between drops
with s hair-dryer until the desired volume had.been spotted. volumes
of 254001.11. were applied with niicropipettes attached to a 1 cc.
syringe. The spotted.papers were then developed in the solvent by a
descending technique in which the entire system was contained in a
12 X 2h inch.batteqy Jar covered.with a glass plate. After develop-
ing, the chromatograms were allowed to dry in a hood and then examined
for various types of compounds.

If any differences were noted on the one-dimensional chromatograms
or if better resolution of the components observed was desired a two-
dimensional chromatogram.was prepared. In all cases the solvents used
were phenol-water in a ratio of four to one and n—butanol-acetic acid-
water in a ratio of four to one to five. The order of solvents was
changed after several runs so that the phenckwater was the last solvent
making the front easier to see in the chromatographic cabinet.

Sheets of thtm n.No. 1 filter paper, 18 l/h X 22 1/2 inches were
spotted near one corner in the same way as used for the one-dimensional
chromatograms. Development of the sheets took place by a descending
technique in closed chromatocabs in.which the paper and atmosphere had
been allowed to equilibrate with the solvent vapors for 30 minutes before
the solvent was added to the trays holding the paper. The developed
chromatograms were thor oughly dried and examined in the same manner
as the one-dimensional chromatograms.

Both types of chromatograms were sprayed with the reagents neces«

sary to produce colored spots with certain types of compounds. The

18
reagents used are listed below;

The Sakaguchi Spray for the guanidine group.

Ammonical silver nitrate for reducing sugars.

p-Dimethylaminobenzaldehyde for indoles and arylamines.

Aniline-phthalic acid for simple sugars.

o-Phenylenediamine for dt-keto acids.

Diazosulfanilic acid for imidazoles.

Ninhydrin for amino acids.

In addition the chromatograms were examined under ultra—violet light
for the presence of any absorbing or flourescent compounds.

Two different solvents were used to develop the one-dimensional
chromatograms. Ethanol-ammonia—water (953115) was used because of its
usefulness in the chromatography of sugar phosphates. However, when
chromatograms developed in this solvent were sprayed with the various
color reagents, the only color produced was at the origin. This was
true even when a molybdate spray for phosphates was used. Therefore,
no results were obtained with this solvent.

The other solvent used, n—butanol-acetic acid4water (hiltl), is
a general solvent capable of resolving almost any type of compound to
some extent. The results which follow were observed with this as the
develOping solvent.

Nearly all of the reagents produced colored spots or streaks with
the aqueous incubation solutions but these were impossible to interpret
without better resolution. The cL—keto acid spray gave spots of about
equal size on chromatograms of both the arginine-inhibited sample and

the control at a Rf value of .28. Since the Spots were of equal size

19
no further characterization of it was attempted. The ether extracts
of the aqueous solutions gave no color with any of these sprays nor did
they show any spots under ultra-violet light. The chromatograms of the
trichloroacetic acid extracts gave colored streaks when sprayed with
ninhydrin, Sakaguchi, diazosulfanilic acid and.the prdimethylaminobenz-
aldehyde sprays and also fluoresced at the origin under ultra~violet
light.

Two-dimensional chromatograms were then prepared of the aqueous
solutions and the trichloroacetic acid extracts of the mycelium. The
aniline-phthalic acid spray showed two spots which were more intense
on chromatOQrams of the aqueous incubation solution containing arginine
than on chromatograms of the control, however, those turned out to be
due to fructose and glucose present as the result of inadequate rinsing
of the mycelia during the transfer from the medium to the incubation
solutions. In addition, a bromophenol blue reagent for detecting acids
and a molybdic acid reagent for detecting phosphates were sprayed on
the two-dimensional chromatograms but again the spots produced.were of
equal size and intensity and so no further investigations were made
along these lines. The other reagents which.produced colored spots or
streaks on the one—dimensional chromatograms were also sprayed on the
two—dimensional chromatograms, but with the exception of ninhydrin,
either no spots appeared or those spots which did appear were of equal
size on.both the control and sample chromatograms.

Many spots sppeared.when the chromatograms of'both the acid ex-
tracts of the mycelium.and the incubation solutions were sprayed with
ninhydrin. The largest spot which appeared.was one with Rf values

corresponding to known values for alanine. Another prominent spot was

20
in the area expected for the dicarboxylic amino acids. The "alanine"
spot was particularly interesting because of its relation to the pro-
posed pathway of pyrimidine biosynthesis and also because of the rela-
tively large amount present.

In order to determine if the spot assumed to be alanine was due
to the ot-orB-isomer, the area of the spot was cut out of an unsprayed
chromatogram and eluted with water. The resulting solution was rechro~
matographed onone-dimensional strips and again sprayed with ninhydrin.

The results as given in Table VI indicate the compound to be oL-alanine.

Table VI
IDEE‘ETIFICATION OF A NIWN-POSIHVE COMPOUND

 

 

Compound Color with Rf in RI in
Nimydrin BuOH-HAc-H20 Phenol-H20
Unknown ' pink , 0.28 0.58
(IL-alanine pink 0.29 0.59
[3 ~alanine blue-pink 0.33 0.60
d-aminobutyric pink 0.10 0.69
acid '

 

Alanine was by far the largest amino acid present in the incuba-
tion solution but the amounts were the sense in both the arginine-in-
hibited sample and the control. It was also a major component of the
trichloroacetic acid extract of the medium, but of a concentration
about equal to the concentration of the dicarboxylic amino acid present.

In one case a ninhydrin—positive spot having an Rf value of 0.142 T
in butanol-acetic acid-water and 0.07 in phenol-water appeared on
chromatograms of the incubation solution containing arginine and not

on chromatograms of the control. However, when an attempt to duplicate

21
the results was nude, the spot did not appear. It was tentatively con- V
eluded to be due to the presence of ornithine, which has similar R!

values, resulting from arginine degradation.

garments on the Arginins Effect
w um op onate- - -

Materials

The sodium propiomte-Z-C“ was obtained from the Volk Radiochem-
icsls Company having a stated activity of 0.5 millicuries per 21.5 mg.
The other compounds used were the same as those used in the previous
experiments. The X-ray run was from the Eastmn Kodak Germany.

Paper Chromatogrspw

No myceiial pads growing on medium supplemented with sodimn
propionate were transferred aseptically with rinsing to two sterile
Petri dishes containing 10 ml. of I solution of 2.6 mg. at sodium
propionate-24:“ and unlabeled sodium propionate so that the total
activity of the solution was about 0.01 so. To one Petri dish was
also added 1 mg. of L-srginins hydrochloride. The pads were incubated
for four hours and then filtered by suction directly into 50 ml.
bsalmrs by use of s all hell Jar. The walls were rinsed and
placed in 25 ml. of acetone for one hour, filtered and placed in a
desiccator to dry.

The total volume of the medium and washings was about 15 ml. in
both cases. This was divided into equal parts. One part was first
acidified to pH 1 with 111. hydrochloric acid, extracted three times

22
with 5 ml. of ether and finally evaporated to dryness and taken up with
1 ml. of water.

One milliliter of the other half of the media was poured into a
5 ml. beaker, covered and placed in the refrigerator. The remaining
portion was evaporated to dryness with a stream of dry air, and taken
up with 1 ml. of water. i

The acid extracts of the wcelia were prepared in the same manmr
as in the previous experiment. In an attempt to recover any volatile
constituents that might be present in the incubation solution, the un-
treated sample and control and the ether-extracts were applied to oner-
dime‘nsional chromtograms in 50 pl and 100 pl quantities and immediately
placed in the chromtography Jars before the paper! was dry. The papers
were developed in phenol-water (hcl), then cut into 1 1/2 inch-vim
strips and the radioactivity counted on a, Forro chromatograph scanner
coupled to a Nuclear-Chicago Model 1620 A ratemeter and an Easterline-
Angus graphic recorder. It was necessary to dry the chromatograms
before counting so that any volatile compounds were possibly lost in
spite of the origiml precautions.

Strip chromatograms were also prepared from the ether-extracted
incubation solutions and the trichloroacetic acid extracts and a
graphic record made of the radioactive spots present.

Tim-dimensional chromatograms were prepared of the incubation
solutions and of the acid extracts of the wcelia. The papers were
again deve10ped in phenol-mater and butanol-acetic acid-water and
sprayed with ninhydrin. The ninhydrin-positive spots for tie arginine-
inhibited sample and the control were again equivalent, but a few of

23
these spots were counted to determine the amount of activity being in-
corporated into the mold components. A.Geiger tube attached to a Nuclear-
Chicago Model 190 ultrascaler was used.for counting the spots directly

from the paper. The results are given in Table VII.

~ Table VII

ACTIVITY or some NINHYDRIN-POSI‘IIVE consensus or N. (33.5st 1298
INCUBATED WITH SODIUM PROPIONATE- 2-c 1'4

 

Trichloroacetic acid extract of mycelium

 

 

 

 

 

 

Rf value of spot Probable Component Activity in countsg/min.
Phenol- BuOH-HAc sample control
H 20 H 20
.59 .23 alanine 13o . 156
.26 .17' (dicarboxylic amino 15h 173
' acid)
Origin -- 3L6 3&5
Background -- 106 106

 

Medium after ether extraction

 

.59 .23 alanine 172 186
.79 182 leucine, isoleucine 123 122

Background -- 10h 10b
.67 .13 arginine 108 ---

 

The strip chromatograms showed high activity at the origin.and at
a few other areas, but no significant differences were seen between
the controls and the samples inhibited with arginine. The activity

present was for the most part of a very low order, especially with the

I acid extracts of the mycelia.

Because of the high amount of activity at the origin, another sol-
vent frequently used to chromatograph sugar phosphates was used in.an
attempt to find a radioactive, phosphorylated intermediate. For this

purpose one—dimensional strip chromatOgrams were developed in methanol-

2h
formic acid-water (8011535 v/v) and the activity determined as before.

again a very low order of activity was recorded except at the origin

and no positive conclusions could be drawn from the record produced.
Radioauwgraphy

Chromatograms of the trichloroacetic acid extracts of the mycelia
were placed against x-ray film and placed in Kodak X-rsy film holders
in the dark. Exposure was allowed to proceed for a period of 10 weeks.
At the end of this period the films were developed in the darkroom of
the Food Science Laboratory, Michigan State University.

Radioautographs were also made of the samples incubated for 10
hours in radioactive sodium propionate-plus-L-mrginine hydrochloride
(0.001 mg.) and of the control containing only the labeled propionate.
The longer incubation period was used to increase the activity of the
mold constituents and the smaller amount of arginine was used to pre-
vent smearing of the chromatograms and also as an attempt to get only
partial inhibition. The incubation solutions of the sample and the
control were acidified, ether extracted and concentrated as before and
spotted on the chromatograms.

A new‘solvent was used since several chromatographic charts of
plant constituents chromatographed in n—butanol-Lpropionic acid-water
and phenol-water were kindly furnished by tr. Kieth Richardson of the
Agricultural Chemistry Department, Hichigan State University.

The new solvent was prepared by mixing 12h6 ml. of n—butanol with
81; ml. of water as solution ”A". Next a solution, "B“, was made by mix-

ing 620 ml. of propionic acid with 790 ml. water. Equal amounts of 'A'

2S
and "B" were stirred together until only one phase remained. More of
solution "B" was added before the two phases merged.

The developed chromatograms were trimmed to fit a 1h X 17 inch
sheet of Kodak X-ray film and placed together with the film in film—
holders. Exposure was for five weeks. The films of the sample and the
control were then developed and compared.

At first glance, no significant difference was noted, however, on
closer inspection s. large spot having an R—alanine value of approx-
imately 1.5 in phenol-water, 1.8 in butanol-propionic acid—water and
2.8 in butanol~acetic acid water was observed to consist of two con-
tiguous spots on the radioautograph of the incubation solution inhibited
by arginine and not on the radioautograph of the control. The cmplete
areas were cut out of both chromtograms, eluted with water and rechro-
matographed on 1 l/2 inch strips of Uhatman No. 1 filter paper using
phenol-water es the solvent. The strips were counted with the Porro
strip scanner and showed two distinct peaks with the strip of the argin-
ine-inhibited sample and only one peak with the control strip. The con-
pounds-have not been identified as yet. The radioautographs of the tri-
chloroacetic acid extracts did not show the same largespot Just men-
tioned, but did show many other spots in comon with the incubation so—
lutions including s. spot for alanine. However, no significant differ-
ence was seen between'the sample and the control.

The nature of the significant component is not known since it was
never seen when the chromtograms were sprayed with the various color-
producing reagents. These reagents are relatively insensitive compared
with the radioautograph process, so the fact that no colored spot was
observed with these reagents does not definitely mean that the specific
type of compounds tested for was absent.

DISCUSSION

To determine the practicality of the use of arginine as a tool
for the isolation of an intermediate in the biosynthetic route from
propionate to pyrimidines in y, 255333.1293, the nature of its inhib-
itory action was investigated. The possibility that the effect of
arginine was due solely to the inhibition of spore germination was
studied since in all previous observations of this inhibitory effect
arginine had.been present in the medium at the time of inoculation
with spores. The results showed.that arginine had the same inhibitory
effect when growth of the mutant was initiated from.mycelial suspen-
sions as when spores were used as the inoculant. This does not provide
conclusive evidence that spore germination was not inhibited since the
lag phase and growth response observed with both types of inoculation
were the same, indicating that growth might actually have started from
spores present in the mycelial suspension rather than from the fragments
of mycelia themselves. However, the fact that arginine definitely in-
hibited growth of intact mycelia as shown in Table III shows that in-
hibition is not solely an effect on Sporulation nor an inhibition of
adaptation at the spore stage.

The results of the growth studies show that much larger concentra-
tions of arginine are needed for inhibition of growing, intact mycelia
than are needed to inhibit initial growth. Probably the most plausable
explanation of this would.be that the larger intact mycelium has the
enzyme systems available for destroying the arginine or of otherwise
removing it from the medium through synthesis of protein and other

mycelial components. This eXplanation would in turn help to explain

27
the presence of the ninhydrin-positive spot assmd to be ornithine
found on chromatograms of the incubation solution containing arginine.

Another possible action of arginine was indicated by the work of
others (25) investigating the inhibitory action of arginine toward
certain histidineless mutants of E. 223333. In this case arginine, in
combination with other amino acids, blocks the transport of histidine
across the mycelial membrane. Evidence that this type of action is not
the reason for the inhibition of §_. 213.23 1298 by arginine is given in
Table VII. The activity of the amino acids extracted with trichloro-
acetic from the wcelium of the mold incubated with labeled propionate
in the presencecf arginine demonstrates clearly that the propionate
was being metabolized. Also, the approximately equal density of the
major spots seen on radioautographs of the trichloroacetic acid extract
of mycelitm incubated with labeled propionate in the presence of argi-
nine and in the absence of arginine shows that propionate is metabolized
to about the same extent in both cases. Thus the assmnption that the
inhibition by arginine is due to the blocking of a specific, vital,
metabolic reaction occurring in the mutant organism seems valid.

Further investigation of the nature of the inhibition of the
growth of the mutant by arginine leads to a consideration of the growth
' characteristics of the mutant on different types of media. The mutant
grows very rapidly on uridine (1h), closely paralleling growth of- the
wild strain on basal medimn. Growth of the mutant on aminobutyrate and
propionate shows a lag period of several days followed by a shorter per-
iod of rapid growth. The lag period is shortened by one to two days by

inoculation with spores from an agar slant containing aminobutyrate as

28
opposed to uracil. These observations led Fairby (28) to conclude that the
new pathway which exists in the mutant grown on propionate or aminobutyrate
is of an adaptive nature and that the process of adaptation is Stimulated
by the presence of increased concentrations of these simple compounds. This
conclusion is further supported by the fact that arginine has no effect on
the growth of the mutant on medium containing uridine or of the wild strain
on basal medium. The interesting observation in the present study of the
growth of the mutant on basal medium may be of importance in confirming the
adaptive nature of this organism. It was very clearly shown that the organ-
ism growing in the blanks was not the wild-type strain since arginine in-
hibited its growth in the same manner as it inhibited the mutant when present
in the medium supplemented with propionate. Prepionate also produced slight
inhibition of this new organism while neither arginine nor propionate had
any inhibitory effect toward growth of the wild-type strain. Therefore, it
seems logical to assume that this new organism is the same mutant 1298 of
31. £323, but has undergone further alteration so that it is now able to
utilise the simple carbon sourcu of the media, sucrose and tartrate, for
the synthesis of pyrimidines.

After a study of all these observations the conclusion was reached
that g. m 1298, because cf a genetic block, is no longer able to syn-
thesise pyrimidines in the manna utilised by the wild-type strain, but,
through adaptation, is able to synthesize pyrimidines by an alternate route
from simple compounds such as propionate. Furthermore, the inhibitory ac—
tion of arginine is caused by the blocking of a specific reaction in this
biosynthetic sequence, and as such, may be properly utilized in the present
study to increase the concentration of the intermediate compounds, which

29
occur prior to the inhibited reaction, in an attempt to gain more in-
formation about this new biosynthetic route.

Many attempts were made to find a compound.present in the mold
inhibited by arginine which was not.present in the control by varying
such conditions as the concentration of arginine or the time of incuba-
tion with.arginine. The concentrations of any intermediates under
these conditions evidently did not increase enough to be detected.by
the various reagents employed or the nature of these intermediates was
quite different from what was expected. The pathway suggested by Boyd
(17) and.presented on page five of the Introduction was used as a guide
to the nature of these possible intermediates. .And, although no direct
evidence was available that arginine acted as a competitive inhibitor,
it seemed very likely'because of its specific nature. If this was the
case, arginine probably would structurally resemble the intermediate
immediately prior to the blocked reaction. Therefore the most reason»
able compound fitting these specifications would.be a derivative of
fig-ureidopropionate. Another possibility would be that the arginine
competes for the enzyme with the carbamyl donor which supposedly coa-
bines with the fl-aiawi-Coi derivative. Because of the high acidity
attained.by the samples during the extractions and chromatography,
hydrolysis of glycosidic, thioester and other similar bonds was likely
and so all combinations of the basic compounds and their derivatives
were looked for as possible spots on chromatograms. No success was
achieved until radioautographs of the incubation solutions were develop—
ed. Many dark spots, corresponding to radioactive compounds on the

chromatograms, were found on the radioautographs. However, only one

30
distinct difference was noted between the radioautograph of the incu-
bation solution which contained arginine in addition to propionate as
compared with the radioautograph of the solution originally containing
only propionate. ’ihis spot was a close neighbor of another mjor spot
found on both radioautographs and might conceivably have been due to a
streak of that compound. However, rechromatography showed that the
spot was indeed a distinct compound. Having definitely established
the presence of another compound by rechromatogaphy there is still
some slight doubt as to its significance because of, among other things,
the small amount of arginine used. However, it is hard to imgine any
other explamtion for the presence of the spot except that it is an
intermediate in prepionate utilization since aseptic techniques were
carefully employed and no contamination was evident when the mold was
harvested. Even if some small amount of bacteria had been introduced
into the incubating sample it seems unlikely that this mch activity
could be incorporated into just one compound without producing other
compo-aids which would be evident on the radioautographs. Mther work
aust now be done to identify the compound and to relate it to the new
route of pyrimidine biosynthesis.

An interesting sidelight to this problem was the finding of large
amounts of alanine which the mold excreted into the propionate solutions
during incubation periods. This proved to be the major radioactive
compound present in the incubation solutions, but not in the trichloro-
acetic acid extracts of the wcelia. This would seem to indicate that
the mutant is capable of converting propionate very quickly into alanine
outside the wcelium. When paper chromtograprw was used to identify

31
alanine it indicated the compound to be oL—alanine, however, when co—
chromatography was employed in which the unknown was spotted together
with otuand fl-alanine, the mixture moved together in both cases, even
in two dimensions. So it is not known with certainity which isomer is
present or if it is a mixture of the two, but the experimental data
favors oL—alanine.

The conversion or prOpionate to fl-alanlhe through the coenzyme A
derivatives of propionic acid and acrylic acid has already been demon-
strated in some microorganisms (26) and has been postulated as being
part or the biosynthetic pathway under study in this thesis. However,
B -alanine did not support growth of this mutant, so that if the compound
was [3 «alanine, its presence in the medium would not seen to be of
significance as a utilizable pyrimidine precursor.

If the alanine is indeed the oL-isomer as indicated or a mixture
of the two isomers, then it is interesting to speculate on the mechan-
ism of its production from prepionate. Alanine is known to arise from
pyruvic acid by a transamination reaction or directly by the addition
of ammonia by the enzyme "balanine dehydrogenase" (27). In support of
this mechanism was the presence of a spot on the radioautographs with
R! values corresponding to those of pyruvic acid.

The extremely rapid conversion to alanine and the possibility of
the presence of both isomers mites one consider the possibility of a
dehydrogenation of propionate and then immediate recombination with
amonia, either specifically or randomly to form oqml amounts or both
isomers. No other evidence to support this possibility is available
and so the conversion to pyruvate and subsequent transamination seems
to be the most plausible mechanism.

. 32

Labeling was also found very high in an amino acid thought to be
either glutamic or aspartic acid. This spot appeared to be, the densest
of all the spots on the radioautograph of the trichloroacetic acid ex-
tract of the mold mycelium. The presence of both of these amino acids
would be expected through operation of the citric acid cycle. The high
amount of labeling of this amino acid in the trichloroacetic acid ex-
tracts as compared to alanine may be significant when more is known

about the metabolism of the mutant.

SUMMARY

1. The action of arginine as a specific inhibitor of pyrimidine bio-
synthesis in E. crassa 1298 was examined with a series of growth and

radioactive tracer experimnts.

2. The preparation of solutions of the constituents of the mold and
their separation by paper chromatOQraphy are described. Samples in
which the mold was inhibited by arginine were compared with samples
from the uninhibited mold, but no significant differences were seen
when the chromatogrsms were sprayed with color-producing reagents.
When labeled propionate was used as the substrate subsequent radio-
autograms of the incubation solution showed the presence of a new com—
pound in the arginine~inhibited sample. The compound has not yet been
identified.

3. me possible significance of this new compound and of other mold

components found is discussed.

1.
2.
3.

9.
10.

11.
12.

13.
111.
15.
16.
17.
18.

19.

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