~-------------------------------------------···--··--------~-- ---------------------~
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California State University, Northridge
i;
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BIOSYNTHESIS OF SERINE IN
''
NEUROSPORA CRASSA
A thesis submitted in partial satisfaction
of the requirement for the degree of
Master of Science in
Biology
by
Fereshteh Khosravi Kline
/
May, 1973
.----,·----··----·-·----·------·--·---·--···--·-------··-··----··-·--·-----1
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The thesis of Fereshteh Khosravi Kline is approved:
Committee Chairman
California State University, Northridge
May, 1973
ii
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ACKNOWLEDGE~lliNTS
1
!
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I wish to extend my sincere appreciation and grat-
j itude to Dr. Joyce Maxwell for her continuous encourage-
:ment, assistance, and guidance on my behalf.
Her dedi-
1
!cation and deep personal involvement in the preparation
i
of this "thesis made this effort possible.
I also wish to extend my deep appreciation to Dr.
· Bianchi and Dr. Spotts for their participation as members
of my graduate committee.
My sincere gratitude goes to Sam Muslin and Elaine
Leboff for their technical assistance.
iii
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--·-·--·----"--------------·-----·-----------·-------··-------,
TABLE of CONTENTS
page
LIST o:f FIGURES •••••••••••••••••••••••••••••••••••••
vi
LIST o:f TABLES • • • • • • • • • • .• • • • • • • • • • • • • • • • • • • • • • • . • • • •
vii
ABST.RACT • ••• • • • • • • • • • • • .. • • • • • • • • • • • • • • • • • • • • • • • • • • • • viii
Chapters
I.
INTRODUCTION •••••••••••••••••••••• , • , ••
1
II.
MATERIALS and METHODS •.•••••••••.••••••
8
Strains . ............................. .
Chemicals . .. , ....•................•...
Maintenance and Growth of Neurospora
Cultures . ............................ .
Genetic Analysis ••••••••••••••••••••••
Extraction of Soluble Enzymes •••••••••
En.z yme As says • • • • • ••••••••••••••••••••
)-Phosphoglyceric Acid Dehydrogenase and Glyceric Acid Dehydrogenase ••.••.•••••••••••••••••
Phosphoserine Transaminase and
Serine Transaminase •••••••••••••
Phosphos~rine
III.
Phosphatase •••••••
RESULTS • ••••••.••••••••
I
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Genetic Analysis of The Mutant Ser-6 ••
Mapping of Ser-6 •••••••.••••.•••
Genetic Instability of Ser-6
Mutant • •.•..•.• , •.•.•.••••••••••
Mapping of Ser-6 on Linkage
I
Group V • ••••••••.•••••.• , •••••••
L__····~··
-iv
17
Studies Concerning the Amino Acid Requirement of Ser-6 ••••••••••••••••.•••
Determination of Serine Requirement •.•.••••••.••••••••••••
Growth of Ser-6 as a Function of
Serine Concentration ••••••••••••
Growth of Ser-6 as a Function
of Time on Vogel's Minimal
Medium and Minimal Medium
Supplemented With Serine ••••••••
The Effect of Inoculum Size on
Utilization of Glycine by
s er- 6 . ...........
a ••••••••••••••
A Comparison of Serine Biosynthetic
Enzymes in Ser-6 and in the Wildtype Culture . ...................... , ..
. )-Phosphoglyceric Acid Dehydrogenase and Glyceric Acid Dehydrogenase •.••••••.•.•••.••••••••
Phosphoserine Transaminase and
Serine Transaminase ••••••.•.•.••
Phosphoserine Phosphatase •••••••
~.
DISCUSSION.. • • . • • • • • • • . • • . • . • • • • • . • • • • • •
48
Genetic Instability of Ser-6 ••••.•••••
The Nature of the Serine Requirement
•
C'
6e
1n
uere e •
e
e e • e
e
e
1
V.
1
1
1
1
1
1
1
1
1
1
1
•
1
1
1
1
1
1
BIBLIOGRAPHY. • • • • • • • • • . • • • • • • . • • • • • • • • • •
v
59
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LIST of' FIGURES
.
Figure
I
page
1.
Pathways of serine biosynthesis f'rom
products of glycolysis •••••••••.••••••••••••••
2.
Pathway of serine biosynthesis f'rom gly-
4
•
7
J. Calculated map of ser-6, his-1 and me-J
on linkage group v ..•...••....•...•...•.•.••••
22
oxyla t e
4.
IJ
••••••••••••••
e •
•
•
•
•
•
•
•
•
•
•
•
•
• •
• •
• •
•
•
•
Published map of his-1 and me-3 on linkage
group V • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • .. • • • • • • • •
22
5.
Growth of' ~er-6 and wild-type Neurospora
as a function of serine concentration •••••••••
27
6.
Time course of' growth of ser-6 on serine ••••••
30
7.
Glyceric acid dehydrogenase and phosphoglyceric aci_d dehydrogenase activities in
the wild-type grown on Vogel's minimal
medium. . . . . . . . . • . . . . . . . . . . . . . . • . . . . . • . • . • . . . • •
.35
Glyceric acid dehydrogenase and phosphoglyceric acid dehydrogenase activities in the
wild-type grown on Vogel's medium supplemented with serine.. . • . • • . • • • • • • . • • . • • • . • . • . • •
37
9.
Glyceric acid dehydrogenase and phosphoglyceric acid dehydrogenase activities in ser-6 ••
39
10.
Serine transaminase and phosphoserine transaminase activities in the wild-type...........
1+1
Serine transaminase and phosphoserine transaminase activities in ser-6 •••••••••••••••••••
43
8.
11.
_12.
13.
Phosphoserine phosphatase activity in the
wild-type.. . . . .. . . . . . . . . . . . . . . . . • . . . . . • • . . . . • . •
45
Phosphoserine phosphatase activity in ser-6 •••
47
vi
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LIST of TABLES
page
Table
1.
Linkage data on random spores isolated
from the cross: ser-6 X his--1, me-
'3.... . . . . . .
20
2.
Linkage data on random spores isolated
from the cross: ser-6 X his-1, me-3 ••••••••••
21
3.
The effect of individual amino acids on
the growth of ser-6 and nutritionally
wild-type al-2; cot-1 •.••.•••.•.
22
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•
•
•
II
;o·J
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····~- ~--~---~-----·--~·
·Vii
.....
,------
-------~------~---·---------l
---·
ABSTRACT
Il
BIOSYNTHESIS OF SERINE
I
IN NEUROSPORA CRASSA
I
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by
Fereshteh Khosravi Kline
Master of Science in Biology
May 197.3
Two pathways for serine biosynthesis in Neurospora
crassa have been reported by Sojka and Garner.
The phos-
phorylated pathway involves )-phosphoglyceric acid, phosphohydroxypyruvic acid, and phosphoserine as intermediates, whereas the non-phosphorylated pathway involves
glyceric acid and hydroxypyruvic acid as intermediates (1).'
The phosphorylated pathway was suggested to be the major
pathway of serine biosynthesis in Neurosnora by these
authors.
of
In this investigation a serine-requiring mutant
Neuros:eor~
crassa was mapped on linkage group V and was
studied in order to determine the nature of its biochemical lesion.
I
i
Each enzyme in the two postulated pathways
was extracted from the mutant strain and compared with the
extract of the corresponding enzyme from the nutritionally·.
wild-type strain of Neurospora crassa from which the mutant was originally derived.
two
path~ays
All enzymes involv:ed in the
were comparable in activity in the mutant and
in the wild-type, indicating that the serine requirement
v.iii
,------·--------·---------------------.
--·~--·--·--~---·-----,
in the mutant does not arise from the loss or alteration
I
of any of the enzymes in these two pathways.
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r--------------------------------~----
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----- --- - ... ···--·---- . _________. __ ··-··· -··-- ---------··--·-_-------------~---------------1
INTRODUCTION
.
.
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!serine Biosynthesis
Synthesis of serine in Neurospora crassa has been
studied by Sojka and Garner (1).
'
i are diagranuned in figure 1.
The postulated pathways
The enzymes active in the
,Phosphorylated pathway are phosphoglyceric acid dehydrogenase, involved in the conversion of 3-phosphoglyceric
acid into 3-phosphohydroxypyruvic acid; phosphoserine
: transaminase, active in transamination of 3-hydroxypyruvic
:acid into 3-phosphoserine; and finally phosphoserine phos-
! phatase which is involved in dephosphorylation of phosphoI
!
; serin6 (figure 1). · The enzymes implicated in the non-phos-•
1
· phorylated pathway are glyceric acid dehydrogenase, in: volved in conversion of glyceric acid to hydroxypyruvic
1
acid; and serine transaminase, active in transamination of
; hydroxypyruvic acid into serine (figure 1).
Because of
·the higher activity of the enzymes involved in the phosphorylated pathway compared to the non-phosphorylated
pathway under the conditions investigated by these authors,
Sojka and Garner suggested that the phosphorylated pathway was the major pathway for serine biosynthesis in
' Neurospora crassa.
The same two pathways for serine bio-
. synthesis were demonstrated originally in animal tissues
(2, 3).
I
In this case the relative contribution by each
pathway for the synthesis of serine varies from tissue to
1
1
2
~~~:::~~~YT~~l~:~~S~~·~~~~~~e~!h~:~~-::~:~~:s-~
j in
higher plants (5).
Studies using isotopic competition
I
land mutants blocked in serine biosynthesis indicated that
i!the
i
phosphorylated pathway is the only major source of
serine in E. coli and
s.
typhimurium (6).
More recent
studies confirming the phosphorylated pathway as the only
significant source of serine were carried out in B. subtilis (7), M. lysodeikticus (8), D. desulfuricans (9), and
H. influenzae (10).
A separate pathway leading to serine from glyoxylate has been suggested in a variety of orgru1isms,
postulated pathway is shown in figure 2.
The
The conversion
of glyoxylate to serine has been shown to be the major
pathway of serine biosynthesis in some animal and pla..nt
tissues (1.1. 12, 13).
Early studies supported the view
that serine precede glycine in the pathway present in
bakerts yeast (14), but later studies using short-term
isotopic labeling indicated that the pathway from glyoxylic acid to serine predominates in this organism (15).
In Neurosnora, Wright reported that a serine-glycine mu•
tant studied by her grew better on glyoxylic acid or glycine than on serine.
She suggested that the biqsynthesis
of serine in Neurospora involves the conversion of glyoxylic acid to glycine and subsequent conversion of glycine to serine (16).
The pathway suggested by Sojka and
Garner clearly contradicts Wright's postulated pathway as
!
Figure 1.
Pathways of serine
biosJ~thesis
from products
The "C 1 .. indicated as a product of serine
metabolism refers to the single carbon fragment used in
. of glycolysis.
:the synthesis of methionine, thymine, and purines.
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,---------··--·-···-~--------··--·-··----·-------·-·-····--·--·--------·-·-··--------··------~----------------,
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It
GLUCOSE
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l
H 0l'OCH yH- COOH
2
2
OH
3-PHOSPHOGLYCERIC ACID
NAtijfNADH
H 0 POCH C -COOH
2 II
2 3
0
3-PHOSPHOHYDROXYPYRUVIC ACID
lJ
transamlna lion
1
HOC H ~H _ COOH
2
OH
GLYCERIC ACID
NA~lf NADH
_c
HOCH
-COOH
2 II
0
HYDROXYPYRUVIC ACID
I ransaminalionlf
H 0 POCH
H _COOH
2 3
2
NH
2
HOCH CH- COOH
2I
NH
2
3_ PHOSPHOSERINE
SERlNE
H
'' Ci' + CH ._ C 00 H
2
I
NH
2
GLYCINE
5
__,__-:._____________._ _ _ _ _ _,_ _ _ _ _ · · -·- . --·-· . -·. - - .-·"'" - ·-·-· ·- ·- -.
1
I
the major source of serine in Neurospora.
.
.
-·----~-------
------,
To support or
refute the pathways suggested by Sojka and Garner, mutants
Irequiring
i
serine were examined to determine whether any of
1
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.
the enzymes utilized in these pathways were deficient or
reduced in activity compared to the wild-type.
A prelim-
: inary study carried out by Sojka indicated that two of the :
four known serineless mutants studied showed no apparent
1
deficiency for any of the enzymes involved in the serine
· biosynthetic pathways postulated by Sojka and Garner (17,
18).
1
The other two mutants both showed alterations in
the transaminases, although they map on separate genetic
sites.
Since the mutants studied were isolated from dif-
ferent wild-types, the difference between the enzyme ac• tivities in the wild-type and the two mutants might have
I
i resulted from differences in genetic background.
I
!
•
~nate
To elim-
the problem of background diversity, Maxwell iso-
: lated five serineless mutants from a single prototrophic
• culture (as described in materials and methods).
One of
these serine-requiring mutants, ser-6 was investigated
extensively as described in this thesis.
The work in this
thesis sought to answer three basic questions:
1.
On
what chromosome is the ser-6 locus located?
2.
What is the nature of the biochemical lesion
in ser-6 that results in its serine requirement?
J.
Does this lesion indicate a major pathway for
serine biosynthesis in Neurospora?
I
j
Figure 2.
Serine biosynthesis from glyoxylate.
Glyoxy-
• late may arise from intermediates in the tricarboxylic
1
acid (TCA) cycle, via the reaction sequence known as the
"glyoxylate shunt".
Isocitric acid is shunted from the
TCA cycle by isocitrate lyase, which splits isocitric
: acid to form succinic acid and glyoxylic acid.
Al terna-
i tively, glyoxyla te may arise from glycolic acid, which is
i
' formed from carbohydrate metabolism.
l
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.. ~
. ··-" ·--··. -·
l
--~-
I
----·--~
-------·------·---------
CITRIC
ACID
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HOCHCOOH
I
I
CHCOOH
I
CH COOH
2
I
ISOCITRIC ACID
HOOC .CH CH COOH
2 2
SUCCINIC ACID
HOCH COOH _ __.,. OCH_COOH
2
GLYCOLIC ACID
GLYOXYLIC ACID
NH3
transamination
NH CH COOH
2
2
GLYCINE
-~~
HOCH CHCOOH
.
2 I
NH
2
SERINE
'
L--·-·-··--·-·--
I
MATERIALS AND METHODS
II
!Strains
I
I
A nutritionally wild type strain of
I;crassa,
I
.
Neuros~ora
strain C102-15300-4-2A, al-2; cot-1 provided by
'
/Mary B. Mitchell was used during these studies.
II
Hereafter :
1
)this culture will be referred to simply as wild-type.
l
;
·, Ser-6 (JBM 4-13) which is used extensively during these
i
studies was isolated by Dr. Joyce B. Maxwell and Mr. Paul
West.
Nutritionally wild type conidia from al-2; cot-1
were irradiated with ultraviolet light to twenty per cent
survival (two minutes).
ing
~-6,
Serine requiring mutants, includ-
were isolated after filtration enrichment {19).
For genetic studies, the strains used were multiple
mutant stock B56; KH2{r); P829;
~'
bal; acr-2;
~
(FGSC
#1540), C102(t); 37401; Y30549y; C86; ~· cot-1; inos;
Xlo-1r nt (FGSC #333), 33933, 37401, Y152 M105, a, J-y:s-1,
inos, his-6 (FGSC #1536), and K744, 361-4,
(FGSC #780).
~·
his-1, me-3
The phenotypic characters of these loci are
described in the table below:
Locus
PhenotyPe
white pigmented conidia
stock grows colonially at temperatures above
300 C.
bal
restricted growth on agar as a hemispheric
colony
acr-2
acriflavin resistant
8
9
~~
c,aroteno.i_d.___deiic"fent-ln mycefia;exc-ep"t at--,
low te.mperature
·
l
inositol requiring stock
Il
I
lnos
ylo-1
yellow pigmented conidia
requires nicotinamide
I
nt
lys-1
I:
his-6
requires histidine
me-3
methionine requiring stock
requires lysine
, Chemicals
Uniformly labeled C14
o(
-ketoglutarate ( 14.3 me/
mmole) was obtained from Amersham Searle Corporation.
To
assure stability of the labile keto acid, the sample was
taken up into steri;te water and dispensed into aliquots
containing enough material to carry out single experiments.
The samples were quick frozen in liquid nitrogen
and stored in -26° C freezer.
Nonlabeled compounds used in these studies were of
reagent grade quality.
Dowex SOW-XB, 200-400 mesh, used for hydrolysis of
hydroxypyruvic acid phosphate and for isolation of glutamic acid from the phosphoserine transaminase reaction
mixture, was obtained-from J. T. Baker Chemical Company.
Before use, the reagent was washed with 2N NaOH, 10 ml/gm
resin.
Then this resin was rinsed with distilled water
the eluant pH was approximately neutral.
Next the
resin was washed with 4N HCl, 10 ml/gm, followed by a dis- '
tilled water rinse.
These resins were used in the H+ form.
1.0
,-----·---------~------·--·----·-~--··----·--- ----------·~-
I_
·--------·----------·-----·
Hydroxypyruvic acid phosphate dimethyl ketal was
!hydrolyzed using the following procedure.
Twenty mg of
salt was dissolved in 2 ml of distilled water.
ml ·of dry Dowex
About 0.4
50 in the hydrogen form was added and
swirled for 60 seconds.
This sqlution was filtered by
suction into a small flask.
This flask was closed tightly
and left in an oven at 40° C for four days.
After four
days the solution was neutralized using an appropriate
amount of sodium bicarbonate.
Biogel P 10 was obtained from Bio Rad Laboratories.
To prepare beads for the column, the dry beads were
stirred into potassium phosphate buffer (pH 8), using 16
ml of liquid for eac;::h gram of dry gel.
allowed to swell overnight.
pack the column.
The beads were
These beads were used to
The column was equilibrated with po-
tassium phosphate buffer at 4° C before it was used.
The scintillation fluid used was prepared from
Spectrafluor PPO-POPOP concentrated liquid scintillator
from Amersham Searle.
Prepared as directed by the manu-
facturer, the scintillation fluid contained 4
gm
PPO and
50 mg POPOP per liter of toluene.
Maintenance and Growth of Neurospora Cultures
Vegetative cultures were maintained either on agar
slants of Horowitz complete medium (20) or Vogel's minimal
N (21) supplemented as required by the particular
strain used.
Stock cultures were kept in a refrigerator
11
~transf~rr;d ..to f'~esh me-diu~-b~f~·re use.
!
·--------
For most growth experiments, cultures were grovm
!without shaking at room temperature in 125 ml Erlenmeyer
i
!flasks containing 20 ml of Vogel's minimal medium Nand
!two per cent (w/v) sucrose.
Each flask was inoculated
)with three drops of conidial suspension.
For determina-
1
!tion of dry weight, the mycelial pads were fished from
I
ithe medium with a spatula after four days of growth,
:blotted dry between sheets of' paper towels, and left overnight at room temperature to dry.
The cultures used for the extraction of' the enzymes
reportedly involved in the synthesis of serine were grown
in Vogel's minimal medium containing two per cent (w/v)
sucrose.
A final
concentra~ion
of 0.4 mg/ml serine was
added for serine requiring cultures.
Fifty ml of' this
medium was used to wash most of the conidia from a five
day old culture grown on 20 ml solid complete medium in
a 125 ml Erlenmeyer flask.
The conidial suspension was
filtered through glass wool before using it to inoculate
700 ml of Vogel's minimal medium contained in a low form
culture flask.
These cultures were harvested after forty
eight hours growth on a shaker bath at 25°
c.
Genetic Analysis
All crosses were done on Westergaard-Mitchell me(22) containing two per cent (w/v) sucrose and supas required by strains used ln the cross.
----·---,
-·------------~----·-----·---·---·--··-------------------
Crosses were prepared by coinoculation of agar plates with
appropriate conidia.
Extraction of Soluble Enzymes
Mycelia were harvested on a Buchner funnel with
suction.
The pads were blotted dry between sheets of
paper towels,
These pads were weighed and then ground
with washed sea sand in an ice cold mortar and pestle.
Glyceric acid dehydrogenase and phosphoglyceric
'acid dehydrogenase were extracted from the broken cells
with five volumes of 0,01 M Tris-HCl, pH 7.5.
The crude
extracts were centrifuged at 12,350 g for 15 minutes.
The supernatant then was centrifuged at 100,000 g for one
hour in a model-L ultracentrifuge.
This supernatant was
assayed for dehydrogenase activity.
Phosphoserine phosphatase was extracted from ground
mycelium with five to ten volumes of 0.01 M Tris-HCl, pH
7.5.
The crude extracts were centrifuged at 12,350 g for
15 minutes.
The supernatant was precipitated with solid
(NH4)2 S04 added to seventy per cent saturation.
This
solution was left on ice for 30 minutes and then centrifuged at 12,350 g for 15 minutes.
The pellet was rinsed
with buffer and suspended in 2 ml of 0.01 M Tris-HCl,
pH 7.5.
Phosphoserine transaminase and serine transaminase
were extracted from ground mycelium with five volumes of
0.01 M potassium phosphate buffer, pH 8,
The mixture
1
1}
c-;;~t;.i.fug;d-;;.t -i-2;).56
g f~r i5 ;..i_;:;,;:;;;,-;:-- Tb.-;-.;;;:pe-rnat.m:;;----~
was then chromatographed on a Biogel P 10 column (2.0 em
,~
x 17 em) at 4° C.
I
The protein peak eluted with 0. 01 M
potassium phosphate
bu~~er
was used for assay.
I
Enzyme As says
I
l-Phosphoglyceric Acid Dehydrogenase and Glyceric
Acid Dehydrogenase
Activity
o~
3-phosphoglyceric acid dehydrogenase
was measured by the disappearance
o~
reduced nicotinamide
adenine dinucleotide (NADH) according to the method of
'
Umbarger et al. (6).
--
[
D-Glyceric acid dehydrogenase was
: assayed by replacing hydroxypyruvic acid for phosphohy,
' droxypyruvic in the reaction mixture.
The reaction mix-
, ture contained 600 ~ moles of Tris-HCl
buf~er
(pH 8. 7),
• 4 I' moles of' MgC1 2 , 0.4 ft moles o~ NADH, and 0.6 fo moles
i of substrate in a. volume o~ 3.2 ml.
Approximately 1.0 mg
i
; o~ protein from a crude extract was used in the reaction
. mixture.
Protein concentration was determined using the
i Biuret test (23).
Oxidation of NADH was followed spectro-
' photometrically in a Beckman DB-G recording spectrophoto- ·
meter at 340 m~ •
Results are shown in fl mole . NADH oxi-
dized per mg protein per hour in the presence
o~
added
substrate minus the background activity in the same units.
Phosphoserine Transaminase and Serine Transaminase
Phosphoserine transaminase and serine transaminase
were assayed using Sojka and Garner's method (1).
The
'--
... - .. -----·----
!
'
--~,-·
Ireaction
r::-------·------------------------·-----------------------~--------1
i
was carried out in the reverse of the serine bio- '
synthetic direction with either L-serine or L-phosphoserine added as the amino donor, and c14 ell-ketoglutarate
I
II as
tl'1e amino acceptor.
The amount of labeled glutamic
lacid formed in this reaction was used as a measure of
.
.
The reaction mixture contained 500
li t ransam~nat~on.
I
I
'
1
moles potassium phosphate buffer, pH 8; 20 p moles L-
.: serine (or 40
moles DL-0 phosphoserine); 20 mg pyridoxal
'
14
phosphate; and 0.06 ~moles of C
«-ketoglutarate {spe7
;cific activity 3.14 x 10 c.p.m/)(mole). This mixture
11
!was incubated at 26° C for five minutes before the reac;
1
tion was begun by adding about 5 mg of protein from a
;crude extract that had been passed through a Biogel P10
i
·column_
Total volume of the reaction mixture was 5 ml.
The reaction was stopped by adding a one ml aliquot of
the reaction mixture to 0.1 ml of trichloroacetic acid.
i Samples were taken at different time intervals.
The pro-
tein precipitated by the trichloroacetic acid was pelleted
I
by centrifugation.
A 0.75 ml sample of the supernatant
was applied to a small Dowex 50 W-X 8, 200-400 mesh, col.
+
umn ~n H form. This column was then rinsed with 20 ml
of distilled water.
NH 0H.
4
Amino acid was eluted with 8 ml 6N
These collected samples were evaporated to dryness
in a vacuum desiccator.
; 0.35
The dried sample was dissolved in
ml of distilled water.
A 0.1 ml sample of this so-
l lution was applied to a small circle of Whatman #2 filter
: paper and evaporated to dryness in front of a heat lamp.
I
15
r----------·-···-·-------·----·--·... -··-·---------·- -- ----- . ____ . ______ -·- --· ---·---- . . --- -.- ·-·-·-- 1
The paper was put into a scintillation
v-ia.T-;·-io-mi
oF·--·----~
scintillation fluid was added, and the sample was counted j
in a liquid scintillation counter, model OJT004A Kobe In-
dustrial Corporation.
m~
Enzyme activity is expressed as
moles glutamic acid formed per mg protein per hour.
The relationship between c.p.m.
and~
moles glutamic acid
was estimated by using known amount of the uniformly la; beled
acid precursor and determining the
~-ketoglutaric
c.p.m. obtained under the conditions of this experiment.
Phosphoserine Phosphatase
Phosphoserine phosphatase was assayed by determin: ing the amount of inorganic phosphate formed from phos: phoserine in the presence of enzyme by the method described by Ames (24).
The reaction mixture contained 200
;){ moles of Tris-HCl, pH 7. 5, 50 tt moles Mg Cl2, 20 /<- moles
of DL- phosphoserine, and approximately 1. 5 mg of protein.
in a final volume of 4 ml.
The reaction mixture was incu-
: bated at 26° C b~fore the reaction was begun.
tion was started by addition of protein.
The reac-
This reaction
was stopped by adding 0.7 ml of the reaction mixture to
2.3 ml phosphatase mix (the mix contains one part 10%
ascorbic acid ru1d six parts 0.42 per cent ammonium molybdate. 4H 20 in 1 N H2S04). This solution was incubated
• for 20 minutes at 45° c. The absorbance of the mixture
was read at 800
m~
in a Beckman DB-G spectrophotometer.
Enzyme activity is expressed
as~
moles phosphate formed
1
16
r-·---------------- "·-----···--------..--- ........... -----· . . -·- .... _-·-----------------------------------..-·
per mg protein per hour.
Genetic Analysis of The Mutant Ser-6
Mapping of Ser-6
To determine the location of ser-6, the mutant was
,crossed with two strains; B56; Kh2 (r); P829;
~;
bal;
. acr-2; we and C102; 37401; Y305J9y; C86; a, cot-1; inos;
ylo-1; nt.
Random spores from these crosses were spread
. on four per cent agar plates.
Individual spores were iso-
;lated onto small slants containing supplemented medium.
• These cultures were heat shocked by placing the tubes in
! a 60° C water-bath for about one hour.
Preliminary re-
sults from these crosses indicated that ser-6 might be on
, linkage group
v.
To further locate the locus on linkage
:group V, ser-6 was crossed with lys-1; inos; his-6.
'
Be-
'cause of the peculiar results of this cross which are de: scribed below, another cross was performed between ser-6
• and his-1, me-J.
Genetic Instability of the Ser-6 Mutant
During the analysis of the cross
invo.Lv~ng
ser-6
i
and lys-1, inos, his-6, the frequency of wild-type progeny I
'
far exceeded the expected value based on reciprocal progeny.
At first, this feature was considered to be the re-
sult of either contamination of crosses by a wild-type
Neurospora or contamination during transfer of conidia to
different media due to poor technique.
17
But several related
f"observat-i0.i18--ieci_t_o_
·z.e-..:evaiU:ation-·oi___th.18___1ligii--wTICi:-=t-Y.Pe -·--1
~~requency as a property o~ mutant
!crosses o~ ser-6 and lys-1, inos,
ser-6
itsel~.
Fresh
his-6 performed with
l
I
I
!
jcareful attention to prevent contamination still showed
l
!a high frequency of wild-type.
Also other individuals
I
[independently evaluated this cross and obtained similar
·results.
Poor technique in transferring was eliminated
:as the responsible factor.
A separate cross involving
ser-6 and his-1, me-3 again showed a disproportionately
·high frequency of wild-type.
An attempt to find a pattern.
! to the results which might explain the cause led to the
; observation that a higher frequency of wild-type progeny
was obtained from crosses involving ser-6 when spore isolates were analysed later than five to seven days after
. germination.
Also in the case of vegetative reisolates
of the original
ser-~i
occur upor1 standing.
mutant reversion to wild-type would
If conidia from these wild-type re-
. vertants were suspended in water and spread on serinesupplemented minimal agar plates and incubated for several
days, it was possible to pick up individual serine-requiring colonies.
These observations suggest that ser-6 has
a high frequency of spontaneous reversion.
Mapping of
~i
on Linkage Group V
Most of the data from the cross involving ser-6 and
lys-1, inos, his-6 were collected when the cause of the
high number of phenotypically wild-type progeny was unknown.
Large numbers of progeny were transferred when the
19
..
,----------------------~---------·-·-·-··--···---···-···--·-··········-~-···-··--·····-··-----------,
isolates were over seven days old.
Jused for mapping the ser-6 locus,
lhis-1,
'
~e-3
These data are not
The data from ser-6 and
crosses can be seen in table 1.
From these
j
l
!
i
jdata only those results obtained from spores transferred
j
:between one to five days after germination are used for
!mapping ser-6.
1
These data are given in table 2.
'
i culated map is shown on page 22.•
The cal-
~-·---
- - - - -.. -•·-"~""-•-•~·--·~ --· ••- • •~ ••---•••
.-,.---·---·---••w••·~--·-••·-••••- •••••-···~--,
- ....- ......
·-·····-·-···-···--·-···-·····--·-· ......
...,_,.,,~---••
__________,
..
I
iTable 1.
1
Linkage data on random spores isolated from the cross:
ser-6 X his-1, me-3
I
These data were collected from progeny transferred between one to fifteen days after
:spore germination.
------------------------------------------------------------1
recombination
I
zygote
genotype
his-1
me-3
parental
combination
single
region I
single
region II
double
region I+II
I
total
I
%germination!
I
----------------------------------------------------------------------1I
+
255
10
5
2
549
201
29
45
2
89-95
+
+
ser-6
I
I
!
region I
region II
----------------------------------------------------------------------------------------------------1
!\)
9
<t·
.
.Table 2.
·----~-~--·---·---------------·-- ·········---~---
. '.
----·-·-·-----
--··· ···-- . - ··--··
I
Linkage data on random spores isolated from the cross:
I
l
I
ser-6 X his-1, me-3.
'These data were collected from progeny transferred between one to five days after germ:ination.
recombination
zygote
genotype
his-1
me-3
+
single
region II
I
parental
combination
singie
region I
108
3
2
0
254
120
13
7
1
89-95
double
region I+II
total
. 1
%germination 1
-------------~~--------------------------------:_________~::~-+
+
region I
-
ser-6
region II
·-···-----~~---~-----~~-"--··----·-------------·--·-·-"···-----~-~-
.,_________
·------ ..
--~----·
- -----·· ·- ·-·· --
-~-------·-·-···
----------------
·----- -------------
------
I
!
I
I
I
I
_____ .JI
N
!-'--"
22
r-------~--··--···--·-··-~ -~----·-·--·-·--·------·- -·---·---------------------~----- ·-·----~---·-·-
. ----------·--------
~
I
! Figure
3.
Calculated map based on table 2.
his-1
ser-6
.___ _ _ 6.6
.___ _ _ _ _
Figure 4.
.....~
10.6 ,___ _ _ _ _ _ ___
Published map (25).
me-3
~------- 10.5
---------.J
23
---~------··----·----------·----··------·----------------·---------l
Studies Concerning the Amino Acid Requirement of Ser-6
I
I
I
Determination of Serine Requirement
!
!
1
The mutant ser-6 was isolated by a technique de•
!s~gne
d
to select serine auxotrophs.
To confirm that
jserine was the only amino acid to which ser-6 would rel
ispond, seventeen amino acids were tested individually.
The result shown in table J, indicated that serine is the
only single amino acid which permits significant growth.
Growth of Ser-6 as a Function of Serine Concentration
As shown in table 3, serine at a final concentration of 0.1 mg/ml does not allow ser-6 to grow as well as
the wild-type.
This result.suggests that ser-6 might re-
quire more than 0.1 mg/ml of serine for optimum growth.
The results shown in figure 5t indicates that optimum
growth of ser-6 is obtained at a final concentration of
0.5 mg/ml of serine.
This amount of serine allows the
mutant to grow to the same extent as the wild-type in
three days.
Growth of Ser-6 as a Function of Time on Vogel's
Minimal Medium and J.Vlin~mal Med~um Supplemented with Serine
Dr. Maxwell (18) observed that a serine requiring
; mutant, DW-110, grows with the same rate as wild-type after
a four day lag on minimal medium, whereas growth of the
wild-type or the mutant in the presence of serine begins
1
[
I
,---------·---------------·-----------·-------------·--------------------------------------------------·------,
~ABLE
I
The effect of individual amino acids on the growth
of ser-6 and nutritionally wild-type al-2; cot-1.
!
w~ld-type
'
1
3.
Added amino acid
al-2; cot-1
mg dry wt
ser-6
mg dry wt
54.6
trace
'DL-alanine
56.8
trace
: L-arginine
62.2
trace
L-aspartic acid
50.9
trace
L-cysteine
45.7
trace
; L-cystine
36.3
trace
; glycine
22.9
trace
· L-histidine
42.9
trace
L-isoleucine + L-valine
51.3
trace
L-leucine
50.0
trace
: L-lysine
49.6
trace
; L-rnethionine
44.5
trace
: L-phenylalanine
47.9
trace
! L-proline
trace
18.5
l DL-threonine
55.3
43.2
41.6
L-tryptophan
37.4
trace
L-tyrosine
53.0
trace
: L-val~ne
• •
33.2
trace
i casamino acids
61.9
trace
none
1
i;
I
!i L-serine
i
!
trace
25
. ··---.--..
r·-~
·------~·-------
3
--~-·-·---··~~.
-·
~ --·-·---~-~- ·-·----~
. ·--
-~.-------· --~--------~----
-l
~~TABLE ~u~:::::n:::: grown stationary ~or three days in 20
ml Vogel's minimal medium in 125 ml Erlenmeyer flasks at
! 25° c. Amino acids were added to give a final concentra\
)
!tion of 0.1 mg/ml for L-amino acids, and 0.2 mg/ml for
l DL-amino acids.
i
: cate pads.
All dry weights are averages of tripli-
I
26
:Figure 5.
Growth of ser-6 and wild-type al-2; cot-1 as
·a function of L-serine concentration.
Cultures were grown
:in 125 ml Erlenmeyer flasks containing 20 ml Vogel's med-
; ium and two per cent (w/v) sucrose.
L-serine was added to
a final concentration which ranged from 0.1 mg/ml to 1e0
• mg/ml.
Flasks were left stationary at room temperature
. for three days.
i
pads.
i
All dry weights are averages of duplicate ·
I
I
i
l!
!
2{.
~---
-----------------
-
-·---------·--·-·-·
I
I
-
70 -
....
0
•
•WILD TYPE
oo--0 SER.6
0.2
0.4
o.6
L.SER (m9/ml)
0.8
1.0
28
at two --d:;;.y;:--·Totest "W"h-~th:;r-·s;r.·:K'"l)-eilaves--similar ly , - - growth of the mutant as a function of time was studied.
The results indicated that ser-6 does not grow on Vogel's
minimal medium even up to six days after inoculation.
The
results of growth on Vogel's minimal medium supplemented
with serine are shown in figure 6.
These results indicate
that the growth of ser-6 on minimal medium supplemented
with serine is negligible up to twenty four hours.
By
three days this growth reaches half of the maximum level
which is attained at four days.
The Effect of Inoculum Size on Utilization of Glycine by Ser-b
observed that in several serine requiring mutants, growth
on 0.5
~
mole of glycine at five days shows a linear re-
lationship with inoculum size.
To test whether the same
phenomenon would be observed with ser-6, this mutant was
grown on 0. 5 )'f. mole of glycine with variable inoculum size•.
The result of this experiment indicates that ser-6 cannot
utilize glycine for growth regardless of the inoculum size.
29
r-----------·----·-·-
·Figure 6.
·-···~-·---·--·---·----··
Time course of growth of ser-6 on L-serine.
I
i Cultures were grown on 20 ml Vogel's medium and two per
1
cent (w/v) sucrose in 125 ml Erlenmeyer flasks.
Serine
was added to give a final concentration of 0.4 mg/ml.
1
Triplicate cultures were harvested and their dry weight
determined at one. to six days after inoculation.
50 -
0
-.,
0
40 -
tit
-
E
Q
30 c(
D.
u.
...::z::0
20 -
~
loll
~
>
~
10
-
Q
5
6
TIME AFTER INOCULATION (days)
.
l
-·--·~----~-'-"'
Jl
I~
-------------··-------·--·----·--- --··----·---·----~-------------------,
A Comparison of Serine Biosynthetic Enzymes in Ser-6 and
Wlld-tlEe Culture
I
'
An attempt was made to identify the location of the '
!altered enzyme in ser-6 which would account for its serine:
!requirement.
All enzymes in the two pathways described by
l
!Sojka and Garner (1) were extracted from ser-6 and the
!
lwild-type and their activities were compared.
The ex-
t
!traction and assay of each enzyme was carried out as de-
.i
! scribed by Sojka and Garner.
,
3-Phosphoglyceric Acid Dehydrogenase and Glyceric
jAcid Dehydrogenase
The first enzymatic step in the two parallel pathways involves phosphoglyceric acid dehydrogenase or glyceric acid dehydrogenase.
The activity of these enzyrrtes
were compared in ser-6 and the wild-type.
The results ob-
tained can be seen in figure 7, figure 8, and figure 9.
These results indicate that neither wild-type nor ser-6
when grown on Vogel's minimal medium containing sucrose
and supplemented with serine in the case of ser-6 shows
any demonstrable activity for glyceric acid dehydrogenase.
The phosphoglyceric acid dehydrogenase activity of the two
cultures grown in Vogel's medium containing sucrose and
supplemented with 0,4 mg/ml serine do not differ significantly from one another.
The specific activity of phos-
phoglyceric acid dehydrogenase in ser-6 corrected for the
background activity as measured by a control containing no
hydroxypyruvic acid or phosphohydroxypruvic acid is 2.0
~
.3-2
:Perli~u-rcomp-a:r-ed.-J
moles-of"·-·N:Anli oxid.iz ed.---p-er -·-mg_o_t_p_ro:Eein
to 1, 70 I" moles of NADH oxidized per mg of protein per houri
in the wild-type.
The
speci~ic
activity
o~
the wild-type
1
'
!
grown on Vogel's medium containing sucrose but no serine
is 1.10
hour.
~
moles
o~
NADH oxidized per mg
o~
protein per
This result indicates that the presence
does not inhibit the activity
o~
serine
phosphoglyceric acid de-
o~
hydrogenase.
Phosphoserine Transaminase and Serine Transaminase
As shown in
10, and
~igure
~igure
11, very little
phosphoserine transaminase activity is demonstrable in the
wild-type or ser-6 within 25 minutes.
However; the phos-
phoserine transaminase activity is equal to serine transaminase activity by 60 minutes in both the wild-type and
ser-6.
For the purpose
speci~ic
~rom
o~
o~
comparing the two cultures,
activity with each substrate has been estimated
the readings at ten minutes.
The
speci~ic
activity
-3
serine transaminase in ser-6 is 3.48 X 10 fi moles/mg/
hour as compared to 2. 57 X 10-3 J4- moles/mg/hour in the wild-;
type.
From the summarized results in ~igure 10 and ~igure
11 and
~rom
a comparison
o~
the
speci~ic
activities, it
may be concluded that ser-6 and the wild-type do not
Phosphoserine Phosphatase
The last enzyme investigated was phosphoserine
I
i
1
I
di~fer'
significantly in the enzyme activities measured under the
conditions of this experiment.
i
!
33
phosphatase.
· - - ·---~-~---------~---·------·----
The summarized results o~ the assay are pr~-
sented in figure 12 and
~igure
13.
The activity
o~
the
enzyme is 5.54.11- moles o~ phosphate/mg/ho~r ~or ~r-.2_ com-
I
1 pared to 2.40~ moles o~ phosphate/mg/hour ~or the wild-
I
1
type.
As shown in
~igure
12,
~igure
13, and also by a
I
!comparison o£ the respective activities, ser-6 does not
!
iSeem to be de~icient ~or phosphatase activity under these
Ijexperimental
conditions.
i
i1
In summary, no major
di~~erences
were demonstrable
:between comparable serine biosynthetic enzymes activities
in ser-6 and the wild-type.
I
I
I
L. . . . . . . . . . ._-·- . . . . · - -·-· - -
II
I
I
i
1
Figure 7.
Glyceric acid dehydrogenase (G.A.D.) and_phos-
; phoglyceric acid dehydrogenase (P.G.A.D.) activities in
: the wild-type grown on Vogel's minimal medium.
Enzyme
activity is measured by disappearance of reduced nicotinamide adenine dinucleotide (NADH).
Oxidation of NADH was
followed spectrophotometrically at 340 m I" •
The reaction
; mixture contained approximately 1.0 mg of protein from a
i
crude mycelial extract.
!
I
I
I
0.6 -
i...
e
0.7 -
c
~
M
Q
.
d
o--o
P.G.A.D.
•
• G.A.D.
o---o CONTROL
0.8
2
4
6
Tl ME (minutes)
8
10
Figure 8.
Glyceric acid dehydrogenase (G.A.D.) and phos-
i phoglyceric acid dehydrogenase (P.G.A.D.) activities in
the wild-type grown on Vogel's minimal medium supplemented
; with serine to a final concentration of 0.4 mg/ml.
Enzyme
'activity is measured by disappearance of reduced nicotin, amide adenine dinucleotide (NADH).
1
Oxidation of NADH was
followed spectrophotometrically at )40 m fi .
The reaction ;
mixture contained· approximately 1.0 mg of protein from a
, crude mycelial extract.
I
,---------~------------------------------------
1
-
I
•
(
e
=
~
Q
0.7 -
M
•
•
0
Q
o.s -
0
0
•
•
0
0
P. G.A.D.
G.A.D.
CONTROL
-I
2
4
6
TIME (
minutes )
8
10
J8
r-·----·---~---··--~--------------------·---·
. ··-.. ____.._
·-··--········---------------------------~--
1
!
I
II
i'
Figure 9.
Glyceric acid dehydrogenase (G.A.D.) and phos-
phoglyceric acid dehydrogenase (P.G.A.D.) activities in
ser-6 grown on Vogel's minimal medium supplemented with
:serine to a final concentration of 0.4 mg/ml.
Enzyme
• activity is measured by disappearance of reduced nicotinamide adenine dinucleotide (NADH).
Oxidation of NADH was
. followed spectrophotometrically at 340 m /f. •
The reaction !
'mixture contained approximately 1.0 mg of protein from a
· crude mycelial extract.
,
i
39
~-·----~·---·----
- - _____.,. .
h~--
~-
-·
·-----~-
·-------------------:
0.6 -
~
e
=
c.•
0
0-7
~
M
8
& P.G.A.D.
••--.,.• G.A.D.
0.8 -
oo---oo CONTROL
2
4
TIME (minutes)
6
8
10
---------------.------------------·-·-·--l
Figure 10.
Serine transaminase and phosphoserine transam-
inase activities in the wild-type. Enzyme activity is
measured by the formation of c 14 -glutamic acid by transamination of
·.j
c 14 ~-ketoglutaric acid in the presence of
enzyme and serine or phosphoserine.
The radioactive glu-
tamic acid product was isolated on a small Dowex column
and counted.
Each point on the graph represents the a-
mount of glutamic acid produced in the reaction mixture
containing approximately 5 mg protein.
Results from two
separate experiments have been combined to construct the
curves.
Points obtained from one experiment are indicated
by ftoo-~o ,
..
e---.t> ,
and ·~---1'1:~.
Points obtained from a
second experiment are indicated by
.,.,
•'----•••------~·
and
. I
·I
--..
G)
as
w
z
••
a:
en
w
0
.Q
:::J
0
0
=
c.r:>
:::J
0 .:
&:~
wcn
0
zoa:
~:x:,_
wa..z
.....'o
=
U")
en
..JCO
..
l ]l
=
~
~
G)
~
:::J
c
·-E
~
w
:i:
-
J-
z
-tc
0
co
::;)
0
z
-I
M
-
-I
I
Figure 11.
Serine transaminase and phosphoserine transam-
inase in ser-6. Enzyme activity is measured by the formation of c 14 -glutamic acid by transamination of c 14 ~-ketoglutaric acid.
The radioactive glutamic acid product was
isolated on a small Dowex column and counted.
Each point
on the graph represents the amount of glutamic acid produced in the reaction mixture containing approximately
5 mg of protein.
Results from two separate experiments
have been combined to construct the curves.
tained from one experiment are indicated by
and*~---4~·
Points ob-
"ifl"•--..•·
I
I
I
o----o•~--~·'
Points obtained from a second experiment are
indicated by •'--••, ____., and
I
,...
--..a
i
Ul
z "':1
De "'
Ul :1
c"'
..Q
!
I
=
-~
V)
l
I
I
0 ·-
:c-5.
I
.... a.. ...
i
:
'
z"'O
-0~
I
Gii::CI-
-fg
wa.z
"l.Jo
...10U
•
~
•
l
]]
- c
-=t
----"'"
·e
._,
:1
II:
Ul
~
.....
=
-M
z
0
.....
<
m
::;)
=
v
%
N
N
-·--···
;
·~---···-·~-~----·---·~------~---"·"--.,--.
_,__...,
I
I
. Figure 1).
Phosphoserine phosphatase activity in ser-6.
The reaction mixture contained 2001' moles of Tris-HCl
(pH 7.5), 50}'(. moles of IVIgC1 2 , 10/" moles of phosphoserine,
and approximately 1.5 mg of protein, in a final volume of
• 4 ml.
Phosphoserine was omitted from one control and pro- •
tein from another.
tion of the protein.
The
reactio~
was started by the addi-
At 5, 10 and 20 minutes. 0.7 ml
aliquots were removed and assayed for inorganic phosphate.
The non-phosphoserine containing control gave a reading
i below zero •
45
COMPLETE
MINUS
PROTEIN
.t
.08
-
-...
tn
Cl)
0
E
-06 -
'
......_,
1.&1
~
:X:
.04 -
A.
V2
0
:X:
A.
.02
5
10
TIME (minutes)
15
20
46
I
!
I
I!
Figure 12.
type.
Phosphoserine phosphatase activity in the wild-
The reaction mixture contained 200je moles of Tris-
, HCl (pH 7. 5), 50 I' moles of 1VIgCl2, 10 )" moles of phosphoj
serine, and approximately 1.5 mg of protein, in a final
!volume of 4 ml.
Phosphoserine was omitted from one con-
. trol and protein from another.
·by the addition of the protein.
; 0.7
The reaction was started
At 5, 10 and 20 minutes,
ml aliquots were removed and assayed for inorganic
, phosphate.
The non-phosphoserine containing control gave
. a reading below zero.
j
~
• _,
--·.-•·~
• ·-•·• ·•- , ..., •• •• ~-·---···-···-L)
MINUS PROTEIN
.12
-
.to -
0
CD
0
E
.08
~
___,
.....
~
:t:
.06
A.
Vj
i
0
:z::
D.
.04
-02
5
10
TIME ( {'ninufes)
15
20
,r·--·--·----·--·-·-···-----·-·-----·-- -------·--·-·---------··-··-·--··-----------·----·---
I
DISCUSSION
I
Ii Gene +"
. . 1.c Instability of' Ser-6
I
A high degree of' spontaneous reversion in vegeta-
I
!
· tive cultures of' ser-6 was observed during this study.
Similar phenomena have been observed by other investigators working
with Neurospora.
Giles in a study con-
:cerning the rate of' spontaneous reversion in inositolless
mutants of' Neurospora crassa observed that one inositol
allele called JH5202 reverted about a thousand times more
: frequently than most of' the other alleles which showed a
f
!uniformly low (0.02-0.01 per million) degree of' revert-
! ibility (26). A high degree of' revertibility was also
.observed by Barnett and De Serres (27) in a study involving a Neurospora mutant, number 137 of' the adenine re~quiring
mutant, ad-3B.
The ad-3B (number 137) allele re-
verted to a phenotypically wild-type allele with a fre2
quency of three to f'our per 10 6 conidia, which is 10 to
104 times more frequent than the reversion at other known
ad-3 mutant sites.
The apparent wild-type obtained by
reversion of' ad-3B (number 137) also showed a high degree
of' instability signified by mutation back to ad-2B (number
: 137) f'rom which they originally arose.
Based on their
observations, Barnett and De Serres concluded that the
ad-JB (number 137) site had become very susceptible to
mechanisms of' spontaneous mutation that cause this site
48
rt~-~s~i t~h·-·b~ck-~d forth bet~-;-~;--two -~nstabl~--;.ii~li~--l
forms.
Unfortunately, very little has been reported about
that could account for this kind of spontaneous
mech~~isms
instability.
The suggestion made by Barnett and De
Serres~
to explain the instability in ad-3B (number 137) is that
the original mutation that gave rise to ad-3B (number 137)
from wild-type was a transversion
(AT~,--~~G)
and the in-
. stability observed is restricted to complementary tran' si tional changes (TM------.CG).
If this b.e the case, the two ;
unstable alleles would rarely mutate back to stable state,
because it would require a transversion.
But each un-
stable form could mutate spontaneously by transitional
change to the other at a high frequency.
i
quires that one of the two unstable alleles permits the
i
I synthesis
1
I
1
Their model re-
of a fu~ctional gene product.
This model could
be tested using mutagens that enhance transversion or
transition.
Bausum in a recent study observed a high prote-
I trophic
frequency caused by meiotic reversion in crosses
I involving certain alleles of an isoleucine-valine (iv-1)
mutant (28).
Crosses involving ser-6 also show a higher
prototrophic frequency than expected, but whether this
high frequency is explained solely by vegetative reversion
or whether meiotic reversion may be involved as well is
still unknown.
50
1-The--Na::~:::e~:::: :::::::::::;::s::::~:::-:l
Ithe
nature of the biochemical lesion in ser-6 which re-
lsulted in its serine requirement.
I
I
Sojka and Garner had published evidence for two
j
I
!pathways of serine biosynthesis in Neurospora (1).
If
!
!these
J
pathways were the major pathways of serine biosyn-
thesis in Neurospora, it seemed reasonable that one of the
i
• enzymes involved in one or both of these pathways would
:be defective in ser-6.
Each of the enzymes involved in
!
the published pathways was prepared from ser-6 and its ac-:
tivity compared to that of the comparable enzyme from the
wild-type.
No
majo~
differences were observed between the
activities of these enzymes in ser-6 and the wild-type
that could account for the serine requirement of the ser-6
mutant.
Sojka investigated the serine biosynthetic enzymes
in the three serine-requiring mutants which were available
at the time he carried out his study.
I
Both ser-2 and
ser-J showed ten per cent of the wild-type activity of
phosphoserine transaminase.
Mutant ser-2 showed three
)
I
times the wild-type activity for serine transaminase, while;
!
ger-1 showed a reduction in serine transaminase activity
(17).
Under the reaction conditions used in this study
and Sojka's study, the transaminase activity is rather low
1
{in this paper negligible for phosphoserine transaminase,
serine transaminase activity was 2.57 X 10 3
~ moles
-·--~~---- ·-- ,.-~-3
51
--..·--------·-·-· ----·····-··-·-·--
-~--·
-·---·-----·-······ ···--- -- ----···· ....
·--·~----------------·---------,
glutamic acid formed/mg protein/hour in al-2; cot-1 wildtype).
It should be noted that the three wild-type
j
strains~
investigated by Sojka, Maxwell and this investigator show.
1
significant differences in transaminase activities. Li-1
\
-2
A has phosphoserine transaminase activity of ).04 X 10
!
fi
moles/mg/h. and serine transaminase activity of 2.75 X 10-2
moles/mg/h .. (17)..
I
Strain ST74 A investigated by Maxwell'
shows phosphoserine transaminase activity of 7,.91 X 10 3 ~
_x
moles/mg/h. and no significant serine transaminase activity (18).
The culture al-2r cot-1 studied in this thesis
shows serine transaminase activity of 2.57 X 10 3 ~ moles/
mg/h. and no significant phosphoserine transaminase activity.
The mutants investigated by Sojka came from diver-
gent backgrounds.
Sojka tried to reduce the effect of
heterogeneity in the genetic background of the mutants by
crosses made to wild-type Li-1 A (17), but it is possible
that this effort was not completely successful.
It is
also unknown whether phosphoserine transaminase or serine
transaminase activity is due to a specific transaminase
or to a general transaminase (1).
Sojka found no differ-
ences in the specific activity of any of the serine biosynthetic pathway enzymes in either H605 ser-1 or 0127
ser-1 compared to wild-type (17).
Another serineless mu-
tant studied by Maxwell, ser-4 (called P110 in her thesis
and later changed to DW-110 by the original isolator) also
i
!did not show any alteration of any of the enxymes involved
I
lin the published serine biosynthetic pathways. Thus ser-1
52
r=------..-·------··
····---·--·"'"'"'-•>-• -
1
1
type activities observed for each of the enzymes indicated j
lin serine biosynthesis in the pathways described by Sojka
I' and Garner.
''
Each of these mutants maps on a different
/linkage group indicating that the three mutants involve
i separate
'
genes.
Wagner et al. observed a group of Neurospora iso; leucine-valine requiring mutants which did not show any
:apparent
de~iciency
in the enzymes involved in isoleucine-
,valine biosynthesis (29, 30).
j
In their study enzyme ex-
tracted from the mutant mycelium showed a high activity
compared to the wild-type, despite the evidence from accumulation of
inter~ediates
and feeding experiments that
, the enzymes were inactive in vivo.
They explained this
:phenomenon by suggesting that the enzyme activity within
the cell requires a particular arrangement of the isoleucine-valine biosynthetic enzymes within an aggregate.
The mutants are postulated to be organizational mutants
in the sense that they are able to produce the necessary
complement of enzymes, but are unable to organize them in
vivo for an effective synthesis of the amino acids.
The importance of organization of enzymes has been
emphasized as well by the work of Lynen.
This investi-
gator studied the mechanism of fatty acid SJmthesis from
i
I
···--·- ........... _ _. ..---'-· .... - .........._____ _. ___ ................... --.. ---·-·---·-·-~------------,
and ser-4 are similar to ser-6 with respect to the wild-
malonyl CoA using enzyme extracts from yeast (31).
He
i suggested that the sequence of the reaction was accomplished by a multienzyme complex.
The functional unit of
I
53
_,_.
~·~~----·--------_.........
......-..........---~-~·~----~--
---
-----,
this complex was proposed to consist of sev~n -d~i-f-f-er-~~t
enzymes arranged around a central sulfhydryl group.
l
Re-
peated attempts to split the multienzyme complex of yeast
into its subunits with retention of the individual enzyme
1
!activities
were without ·success (31).
I
Ii
Whether the explanation for the serine requirement
lor ser-6 mutant involves organization of serine biosyn-
1
-
)thetic snzymes into an aggregate is still unknown.
ling for accumulation of intermediates and studying various
'
!fractions in the enzyme preparation (soluble versus sedi1
!mentable material) for the enzyme activity might provide
.
!additional insight into the nature of the ser-6 mutant.
I
J
An alternative explanation for the behavior of
ser-6 could be that in vivo the activity of one or more of
I the serine biosynthetic enzymes is inhibited by some subI stance which is r~moved during preparation of the enzyme
I extracts& An example of this phenomenon is found in the
i
1
work of Suskind and Kurek who studied tryptophan - re-
Iquiring
I for
(td) mutants which are unable to utilize indole
growth and which lack tryptophan synthetase activity
(32).
They found a mutant, td24, which requires try-
ptophan for growth at 25° C, but which grows sl~wly without tryptophan above 30° C, and at this temperature also
forms a slight amount of tryptophan synthetase.
They were
able to obtain highly active tryptophan synthetase from
this mutant grown at 25° C, by suitable fractionation of
crude inactive extracts.
I
i
Search-i
t
J
II
I,
The active enzyme obtained by
54
IthiS :fractionation method was :found to differ from the
r~-_x--~--<
I
~
......... ,--.._~---~~.......-.--~·~-- ~ ·· ·-----~·---·~-~--- ~--··- ···-
-~--·
----- ---- ~-~---~ --~~-·-·--- ----~---------~--
---H--
enzyme obtained from wild-type in its abnormally high sensitivity to inhibition by zinc ions.
Thus it seems that
J
!the mutation has caused this enzyme to become sensitive to
i
!zinc (or some other metal inside the cell whose effect
'
!could be mimiced by zinc) at 25° C.
The process of frac-
tionation in fact removed this metal from the enzyme and
.i hence removed the inhibitor of' the enzyme's activity.
Another possible explanation for the results ob. tained with ser-6 is that the two pathways studied by
1
Sojka and Garner may not be the major pathway of serine
!biosynthesis in Neurospora crassa.
The fact.that this
mutant strain does not grow on glycine, while glycine and
serine are thought to be interchangeable (JJ), suggests
i that glycine might precede serine in the major serine bio-
synthetic pathway.
In that case, ser-6 could be deficient
in serine aldolase which is the enzyme involved in synthe- '
sis of serine from glycine (J4).
Evidence that serine may be derived from glycine in ·
Neurospor~
was given by Sojka (17) and was confirmed by
Maxwell using radioactive glycine (18).
Abelson and Vogel
obtained evidence from labeling studies in
Neurospor~
t'
.
tha t ra d 1oac
·1v1' t y· f rom
c 14-g1 yc1ne
·
· 1ncorpora
·
t ed
1s
: into serine and cysteine (35).
Pitts et al. observed that
;crassa
---
--
!
in rat kidney serine is synthesized from c 14-glycine (J6).
i
De Bciso et al. observed that in baker's yeast the synthe- ;
:sis of serine takes place by condensation of glycine with
55
~~--~;-~:.::~;~b~;;_--;~i t deriv~d·-·f~~~-ci1i:-.::f~~m~te :-Thei-;;:i~~b-l
served that radioactivity from
1
I directly
c 14-glycine is incorporated .
into serine (15).
1
A suggested precursor of glycine is glyoxylic acid.
i
14
In baker • s yeast De Boiso et al. obs~rved that [ 1-c ] glyoxylate and [ 1-c 14) glycolate are converted into serine
and glycine.
Glycolic and glyoxylic ac·id labeled with C14
1
have been shown to be effective precursors of glycine in
animal tissues (11, 12).
!
·
Wright showed that a mutant of
Neurospora crassa which requires glycine or serine also
utilizes glyoxylic acid or glycolic acid.
She suggested
that in the biosynthesis of glycine and serine by Neuro~ora
crassa, glycolic acid can be oxidized to glyoxylic
acid which can then be aminated to glycine (16).
Results
contradicting Wright's suggestion have been reported by
another group of investigators.
Combepine and Turian were
unable to grow ser-1 on glyoxylate and hence concluded
that this pathway must not play a major role in glycine
synthesis in Neurospora (37).
Maxwell determined that the
difference in Wright's and Combepine's results could be
explained by the difference in carbon source used by these
two investigators (18).
Combepine et al. used sucrose in
--
their media and no serine-glycine auxotrophs utilized glyoxylate for growth.
Wright used glycerol, in which case
the seri.ne-glycine auxotroph was able to utilize glyoxylate,.
Maxwell (18) retested mutants studied by Comb~pine and
I
: ....Turian on glycerol and shewed that all of them utilize
L~
56
··-·-------·---------······'"-·-·---· .... -... ---.. . .. .. ........ ·----- --------- --....·------------------------·--1
Ir·-·-·-----·-····-----glyoxylate on this carbon source. This observation indi-
1
cated that the carbon source plays an important role in
the utilization of glyoxylate.
An example of the impor-
tance of carbon source in serine and glycine metabolism
is found in the work of Ulane and Ogur (38) who studied
serine-glycine biosynthesis in Saccharomyces.
They ob-
served that in this organism both the phosphorylated path: way from products of glycolysis and the glyoxylate pathway
: from tricarboxylic acid cycle intermediates were operative.
!
l
The phosphorylated pathway appeared to be the principal
biosynthetic pathway to serine and glycine during growth
on glucose media, while the glyoxylate pathway was the
major pathway
durin~
growth on acetate.
To explain their
results, they suggested that the glyoxylate pathway to
glycine is repressed by growth on glucose¥ specifically,
at isocitrate lyase.
The repression of isocitrate lyase,
which splits isocitric acid (derived from tricarboxylic
acid cycle) to form succinic acid and glyoxylic acid, has
been observed also in Neurospora by Sjogren and Romano
(39).
Flavell has observed derepression of the enzymes
of the glyoxylate shunt after transfer of cultures from
sucrose medium to one in which acetate was used as the
carbon source (40).
So it would appear that on the suc-
rose medium used in the present study tricarboxylic acid
cycle intermediates cannot be utilized as precursors of
'
l glyoxylic acid.
An alternative precursor for glyoxylic
acid could be glycolic acid.
Glycolic acid has been shown
I
I
57
.
rby--~~~-~- i~;~-~t-l-g~t~;~· -t~- --b~. --~~---~-ff;c--ti.;·~---p;;;c u~~-0-;--;:r-gi-y:l
loxylic acid (11, 12, 16).
Tolbert and Cohan studied syn-
lthesis of glycine in barley and wheat leaves. They used
'
lo 14 labeled glycolic acid and found that the major prod1
i
:ucts formed from glycolic acid are glycine and serine (1J).
/Another alternative precursor of glyoxylic acid suggested
'
by Hardy and Quayle is methanol which is utilized by Pseu_\domonas AM1 to synthesize glycine and serine via glyoxy!
late (41).
Whether the major pathway of serine biosynthesis
'
linvolves methanol, glycolic acid, glyoxylic acid, and/or
glycine is unknown.
To get more information-about the
pathway of serine synthesis and the location of the biochemical lesion in ser-6 extensive studies involving metabolism of uniformly labeled methanol, glycolic acid,
glyoxylic acid, glycine and serine in both ser-6 and wildtype will be necessary.
Another explanation for the results obtained with
· ser-6 is that the mutant may not be defective in serine
bios~mthesis
at all, but may be defective in the metabo-
lism of active "01" units.
Serine has been implicated as
the reservoir for "C 1 " units in yeast (15) and may serve
the same function in Neurospora. If the production of
"01" units from other sources were blocked in ser-6,
serine might be channeled into reactions requiring "0 1 "
units,
To test this possibility for ser-6, growth of the
mutant could be evaluated on sodium formate or formaldehyde.
58
,----·-----·-···--·-·-·· ----···---·--·-------·-· ...................................------- .... ---------------·
I.
--·------~
The :fact that ser-1, ser-4, and ser-6 show no alte!'-1
ations in the biosynthetic pathways leading to serine which!
lhave been proposed by Sojka and
Garner suggests that serine i
I
!metabolism in Neurospora is not yet fully understood.
I
!simplest
eA~lanation
The i
for these mutants is that an altern-
iative pathway for serine biosynthesis exits in Neurospora
l
:which remains to be elucidated.
Alternatively these mu-
tants may be defective in their ability to form a functional enzyme aggregate required for serine biosynthesis
or they may be regulatory in nature.
Clearly, additional
study of serine biosynthesis in Neurospora will be necessary to explain these paradoxical mutants.
BIBLIOGRAPHY
I
1.
G. A. Sojka and H. R. Garner. 1967. The serine bio-'
synthetic pathway in Neurospor~ crassa. Biochim.
Biophys. Acta 148, 42.
2.
A. Ichihara and D. M. Greenberg. 1957. Further
studies on the pathway of serine formation from
carbohydrate. J. Biol. Chern. 224, 331.
3.
J. E. Willis and H. J. Sallach. 1962. Evidence for
a mammalian D-glyceric dehydrogenase. J. Biol.
Chern. 221, 910.
4.
D. A. Walsh and H. J. Sallach. 1966. Comparative
studies on the pathways for serine biosynthesis
in animal tissues. J. B:i.ol. Chern. 2l~1, 4068.
5.
J. Hanford and D. Davies. 1958. Formation of phosphoserine from )-phosphoglycerate in higher
plants. Nature 182, 532.
6.
H. E. Umbarger, M. A. Umbarger, and P. M. L. Siu.
1963. Biosynthesis of serine in Escherichia coli ·
and Salmonela tyPhimurium. J. Bacterial • .§.2,--1431.
7.
M. Ponce-De-Leon and L. Pizer. 1972. Serine biosynthesis and its regulation in Bacillus subtilis.
J. Bacterial. 11Q, 895.
8.
J. D. Nelson and H. B. Naylor. 1971. The synthesis
of L-serine by Micrococcus ±xsodeikticus. Can.
J. Microbial. !Zp 73.
---
9.
G. J. Germano and K. E. Anderson.· 1969. Serine
biosynthesis in Desulfovibrio desulfuricans. J.
Bacteriol, 99, 893.
--
10.
L. Pizer, M. Ponce-De-Leon, and J. Michalka. 1969.
Serine biosynthesis and regulation in Haemophilus
influenzae. J. £3aEteriol. 21• 1357.
-
11.
A. Weissbach and D. Sprinson. 1953. The metabolism·
of 2-carbon compounds related to glycine. J.
Biol. Chern. 203, 1023.
12,
s.
Weinhouse and B. Friedmann. 1951. Metabolism of
labeled 2-carbon acids in the intact rat. J.
Biol. Chem. 121, 707.
59
60
----·-·------------···--····---------------··--··--···--------..
----~
N. Tolbert and M. Cohan. 1953. Products formed
from glycolic acid in plants. J. Biol. Chem. 204,1
1
649.
l
---
14.
G. Gilvarg and K. Bloch. 1951. The utilization of
acetic acid for amino acid synthesis in yeast.
J. Biol, Chern. !22• 339.
15.
J. F. De Boiso and A. o. M. Stoppani. 1967, Metabolism of serine and glycine in baker's yeast.
Biochim. Biophys. Acta. 148, 48.
16.
B. E. Wright. 1951. Utilization of glyoxylic acid
and glycolic acid by a Neurospora mutant requiring glycine or serine. Arch. Biochem. JiiophJI:f:i.•
II
!
i
.J!, 332.
G. A. Sojka. 1967. Ph. D. Thesis, Purdue University, Lafayette, Indiana.
18.
J. B. Maxwell. 1970. Synthesis of L-amino acid
oxidase by a serine or glycine-requiring strain
of Neurospora. Ph. D. Thesis, California Institute of Technology. Pasadena, California.
19.
v.
20.
N. H. Horowitz. 19l-1-7. Methionine synthesis in
}:'leurospora, The isolation of cystathionine.
Biol. Chern. ~. 255.
21,
H. J. Vogel.
W. Woodward, J. R. DeZeeuw, and A. M. Srb. 1954.
The separation and isolation of particular biochemical mutants of Neuro~ora crassa by differ- :
ential germination of conJ.dia, followed by filteration and selective plating. Proc. Natl. Acad.
Sci. u. s . .i2_, 193.
-
1956. A convenient growth medium for
Neurospora (medium N). Microbial. Genet. Bull.
I.J,
I
I
j
'-~-'
J.
42.
22.
M. Westergaard and H. K. Mitchell. 1947. Neurospora v. A synthetic medium favoring sexual reproduction. Am. J. Bot • .2i, 573.
23.
A. G. Gornall, C. J. Bardawill, and M. M. David.
1949. Determination of serum protein by means of
the Biuret reaction. J, Biol, Chern. !ZZ, 751.
24.
B. N. Ames, 1966. Methods in Enzr;ology VIII (ed,
s. P. Colowick and N. o. Kaplan~ Academic Press,
p.
115.
62
---·--------·--------·---~---·-·----~---------------1
G. Comb~pine and, G. Turian. ~965. Recherches Sur
i
la b~osyntheses de la glyc~ne chez Neurospora
!
crassa type sauvage et mutants. Path. Microbial. :
2~, 1o1a.
.
-I
J8,
R. · Ulane and M. Ogur. 1972. Genetics and physiologic.al control of serine and glycine biosynthesis
in Saccharomyces. J. Bacterial. !Q2, 34.
39.
R. E. Sjogren and A. H. Romano. 1967. Evidence for
multiple forms of isocitrate lyase in Neurospora
crassa. J. Bacterial. 2l• 16J8.
40.
R. B. Flavell. 1966.
Neurospora crassa.
41.
w.
The glyoxylate cycle in
Heredity. 21, 343.
Hardy and J. R. Quayle. 1971. Aspects of glycine and serine biosynthesis during growth of
Pseudomonas AM1 on c 1 compounds. Biochem. J.
121, 76J.
l
·----------··------·----·--·------·---------
-·-·
·-··-··----------··-
-------
>
------·
·--···
>
--·
---
·---
----------
-----