J. Cell Sci. 7, 531-548 (1970)
Printed in Great Britain
MUTATIONS INDUCED IN AMOEBA PROTEUS
BY THE CARCINOGEN iV-METHYL-JVNITROSOURETHANE
M. J. ORD*
Zoology Department, The University, Southampton, England
SUMMARY
Mutations have been induced in single amoebae by treatment with the powerful mutagen,
iV-methyl-iV-nitrosourethane. Three mutants, which differ visibly from the parent strain to the
extent that individual amoebae are recognizable and change of characteristics from normal to
mutant or mutant to normal can be followed in single cells, are described. These mutant strains
have been cultured in the laboratory, using Tetrahymena feeding, without loss of mutant
characteristics for over 2 years, undergoing some 100 or more cell cycles. Hybrids made
between mutants and the parent strain, using the nuclear transfer technique, are viable and
stable. They showed that in all cases crosses between normal and mutant amoebae retained the
characteristics of the mutant only if the mutant nucleus was present. Though amoebae from
2 of the mutant strains may revert to normal form with increase in temperature or change to a
wheat culturing technique, these 'apparent normal' amoebae resume the mutant form on a
return to the temperature and feeding conditions normally used.
The interaction between mutant and control amoebae was observed in heterokaryons over
periods of up to 6 weeks by cutting off pieces of the heterokaryon cytoplasm at regular intervals
to prevent division while allowing growth. In general the presence of a control nucleus led to
the development of normal characteristics whether the heterologous nuclei were contained in
control or mutant cytoplasm. No antagonistic action occurred between mutant and control
nuclei when sharing the same cytoplasm. Study of the clones formed by the offspring of
heterokaryon divisions showed the mutant and control nuclei unaltered by long periods of
close contact. These experiments indicate the chromatin as the site of action of the mutagen.
A possible mode of action of the mutagen in amoebae, relating to the functional state of the
DNA, is suggested.
INTRODUCTION
Amoeba proteus can differ considerably under natural conditions, and strains which
show variations in characteristics such as sensitivity to irradiation (Kalney, 1969),
ability to attach to the substratum (Lorch, 1969), or tolerance to chemicals such as
streptomycin (Hawkins & Cole, 1965; Kalinina, 1969), neomycin (Hawkins &
Willis, 1969), methionine and ethanol (Yudin, 1961) have been used in inheritance
studies. In general, if these strains differed to any marked degree transfers of nuclei
between them produced few if any clones. In investigations where nuclear transfers
yielded viable clones, selected differences were shown to be dependent on either the
nucleus or the cytoplasm, or both the nucleus and cytoplasm together. The strains
* Member of the Toxicology Research Unit, Medical Research Laboratories, Carshalton,
Surrey, England.
34
c EL
7
532
M. J. Ord
used in this investigation differ in character from any of the above in that the differences are not naturally occurring or selected but are induced in individual amoebae by
the action of a known mutagen, an event which has never before been achieved.
Nuclear transfers between such mutants and the parent strain are viable and no
antagonistic action occurs between mutant and control nuclei when sharing the same
cytoplasm.
Mutants have been produced in Amoeba proteus by treatment with iV-methyl-iVnitrosourethane (MNU), which ranks among the most powerful of known mutagens
(Magee & Barnes, 1967). In this paper 3 particular mutants have been used to investigate 3 principal questions: (1) the site of action of the mutagen and participation of
nucleus and cytoplasm in the change from normal to mutant form; (2) the stability
of the mutant strains; and (3) the effect of mutant and normal nuclei upon each other.
All three mutants, Mini Mutant, SpG Mutant and Pale Mutant, originated from
single amoebae after treatment with 1 x IO~3M MNU and have been cultured in the
laboratory for more than 2 years, undergoing at least 100 cell cycles, without loss of
mutant characteristics. Each differs from the control parent strain in a number of
characteristics apparent in both the nucleus and the cytoplasm. Moreover, each is
visibly different to the extent that individual amoebae are recognizable and change of
characteristics from normal to mutant or mutant to normal can be followed in single
cells.
MATERIALS AND METHODS
Control and Mini Mutant amoebae were mass cultured in large plastic dishes; SpG and
Pale Mutant amoebae (which have poorer attachment and are difficult to see) were mass cultured in glass crystallizing dishes; for experimental work and single cloning, amoebae were
grown in small solid watch glasses. During single cloning, amoebae were followed for 6 cell
cycles in all cases, with samples of each hybrid, and offspring of all heterokaryons, grown to
small mass cultures. The culture fluid was a modified Chalkley's medium (16 g NaCl; o-8 g
NaHCO 3 ; 0-4 g KC1; 0-2 g Na 2 HPO 4 . i2H 2 O; 0-2 g CaHPO 4 and 0-2 g MgCl 3 to 1 1. of distilled water, diluted on use by 5 ml to 1000 ml distilled water), adjusted before use to pH 6-2 +
o-i. Amoebae were fed with Tetrahymena, grown on proteose peptone under sterile conditions
and washed 3 times with Chalkley's solution on a No. 4 sintered glass filter. Large cultures
were fed daily, except Sunday; smaller dishes were fed at each change; all dishes were changed
at least 3 times per week. Unless otherwise stated all amoebae were cultured at 18-19 °C.
Volume measurements were made by drawing the amoebae into a straight-walled pipette
(diameter 62 /*m) and measuring the length of pipette occupied by the amoeba against a mm
scale. The same pipette was used throughout the entire work. Whenever possible volume
measurements were made on division spheres which give a more representative size measurement for a clone. All nuclear transfers were done with a Fonbrune micromanipulator using the
oil chamber technique of Comandon & deFonbrune (1939). Cutting of hybrids and of heterokaryons was carried out free hand at a magnification of x 50 in the watch glasses in which the
amoebae were growing. The change in form of the cytoplasmic triuret crystals (Griffin, 1961)
was used to identify both Pale and SpG Mutants.
Amoebae were examined for birefringent crystals at magnifications of x 70 and x 100 using
a monocular microscope with one Polaroid filter above the condenser and one in the microscope eyepiece. Treatment of amoebae with Af-methyl-iV-nitrosourethane has been described
in detail elsewhere (Ord, 1968a).
Nomenclature for transfer operations and resultant hybrids or heterokaryons is as follows:
The transfer of a Pale Mutant nucleus to control enucleated cytoplasm (PMn->Cc) gives the
hybrid PMnCc; transfer of a Pale Mutant nucleus to a control amoeba (PMn^-Cn Cc) gives
Carcinogen-induced mutations in Amoeba
533
the heterokaryon PMnC,,Cc. Transfer of a control nucleus to SpG Mutant enucleated cytoplasm (Cn->SMC) gives the hybrid Cn SMC; transfer of a control nucleus to an SpG Mutant
amoeba (Cn->SMn SMC) gives the heterokaryon C,,SMnSMc, etc.
RESULTS
Characteristics of mutants
The Mini Mutant (MM) (Fig. 6). This mutant strain came from an amoeba treated
32 h after division (mid G2) with 1 x IO""3M MNU for 25 min (Nov. 1967). Division
after treatment was delayed for 20 days. Once the amoeba began to divide it was
apparent within 4 divisions that the clone being formed consisted of amoebae uniform
in size, but smaller than normal. The clone was grown to mass culture and has now
been kept, with Tetrahymena feeding, for 2 years. Though larger amoebae frequently
Table 1. Some characteristics of normal, mutant and hybrid amoebae
(Average volume for a clone was obtained using approximately 100 division spheres.
Average nuclear long diameter was obtained from measurements of 100 nuclei
using amoebae of mixed ages.)
Average
volume of
division
Strain of
amoebae
spheres,
/tm3
Average
nuclear
Average
long
cell cycle
diameter, length,
/tm
days
Type of cytoplasmic
crystal
X6y and related
strains (controls)
3700000
46
2-2-5
Numerous truncated
tetragonal bipyramidal
crystals; av. size
1-2 /tin; isotropic
Mini Mutant
2300000
37
6
Pale Mutant
3800000
49"5
S
Bipyramidal crystals as in
controls
Square or rectangular
platelets; av. size along
edge 5-10 /tm;
SpG Mutant
3 000 000
43
MMnCc. hybrid
CnMMc hybrid
(3 months
after transfer)
PM,,CC hybrid
2400000
3450000
38
5-6
46-6
2-3
5-6
—
—
5
—
—
2-5
CnPMc hybrid
(3 months
after transfer)
SMnCc hybrid
3000000
44-2
5
CnSMc hybrid
3400000
48-7
25-3
+ birefringence
Crystals as for Pale
Mutant but fewer
As in controls
As in controls
As in Pale Mutant with
+ birefringence
As in controls
As in SpG Mutant with
+ birefringence
As in controls
(3 months
after transfer)
34-2
534
M.J.Ord
appear in the culture dishes these are generally either non-viable, or separate at
division into 4 instead of 2 daughter cells. During the first 6 months after treatment
these amoebae had a cell cycle of near normal length, i.e. 48 h, but this cycle gradually
increased to 6 days. Characters used as markers to identify Mini Mutants are: (1)
size, approximately half parent strain (Table 1); (2) nuclei, smaller than parent
strain (Table 1); (3) tendency of amoebae to adopt a rosette form even in the absence
of food; (4) approximately 3 % binucleates as compared with 0-5% for control
amoebae; and (5) a cell cycle of 6 days.
The Pale Mutant (PM) (Fig. 3). This mutant strain arose from an amoeba treated
48 h after division (end of G2) with 1 x IO~3M MNU for 30 min (Jan. 1967). Treatment delayed division for 21 days and subsequent divisions were very slow with at
least 25 % of amoebae non-viable. By the third division after treatment all amoebae
were very pale, with fewer crystals in the cytoplasm. By 5 months approximately 25 %
of the clone developed a new type of crystal, a large square platelet with average edge
length of 5-10 /tin and + birefringence when viewed between crossed Polaroid filters
(Ord & Bell, 1968). These amoebae were grown to mass culture using Tetrahymena
feeding and have been kept under these conditions for 2-5 years. Characters used as
markers to identify Pale Mutants are: (1) cytoplasmic crystals in the form of square
platelets with + birefringence (Figs. 4, 5); (2) a cell cycle of 5 days; and (3) nuclei
slightly larger than normal and crumpled in shape (Table 1).
The SpG Mutant (SM) (Fig. 7). This mutant strain arose from an amoeba treated
8 h after division (end of S) with 1 x IO~3M MNU for 25 min (Nov. 1967). Division
after treatment was delayed for 18 days and the paleness of the offspring was apparent
from 3 divisions after treatment. The clone grew very slowly with many non-viable
offspring and was kept in small dishes for 5 months, during which time normal cytoplasmic crystals were gradually lost and replaced by a small number of square platelets of the type found in the Pale Mutant. The heavy bodies of the cytoplasm, generally
small and inconspicuous in normal amoebae, became more numerous and larger
with diameters as much as 15 /mi. Because of the lack of crystals these amoebae have
a ghost-like appearance and under the dissecting microscope are visible chiefly due
to food vacuoles which appear as black spots giving the characteristic 'spotty ghost'
appearance.
Characters used as markers to identify SpG Mutants are: (1) transparent appearance
due to the very small number of cytoplasmic crystals; (2) -(-birefringence from these
crystals which are of the square platelet type; (3) large size of heavy bodies; (4) a cell
cycle of 5 days; (5) smaller size (Table 1); and (6) change in membrane as indicated
by probing with a microneedle, by poor attachment to the substratum, and lowered
ability to catch food.
The site of action of the mutagen
Nuclear transfers between normal A. proteus and each of the mutant strains were
used to find the site of action of the mutagen. Table 2 shows the combined results of
these experiments. In all cases crosses between normal and mutant amoebae retained
the characteristics of the mutant only if the mutant nucleus was present.
Carcinogen-induced mutations in Amoeba
535
In Fig. 1 the change in volume of MMnC(. and CHMMC hybrids has been correlated
with their division. In transfer operations of both types there was generally a delay of
4 days before division began. Where a Mini Mutant nucleus was put into the comparatively large volume of control cytoplasm this delay had no accompanying growth,
and during subsequent divisions size decreased rapidly. Where a control nucleus was
Table 2. Results of nuclear transfers between mutant and control amoebae
Final form
mutant
Non-viable
Type of
hybrid
PMnCe
C,,PMC
SMnCc
CnSMc
MM,,C,
C,,MMC
c,,ce
No. of
transfers
No.
85
21
A
6
80
90
17
5°
8
124
90
13
50
9
3
^
/o
24
7'5
18-5
16
10-5
10
6
2
A
f
Final form
normal
*
1
No
%
No.
64
—
76
—
—
73
—.
8i-5
H I
—
—
3
—
89-5
—
—
4
74
—
v
/o
—.
92-5
' —
6
84
2
2
2
—
42
—
—•
81
90
47
94
5
Average
time
required to
complete
change,
weeks
6
2
3-4
7
Number of cell cycles after transfer
Fig. 1. The change in volume which occurred in Mini Mutant and control hybrids.
Volume is represented in units such that 1 unit = 300000 /im3. Each measurement
O) and CnMMc hybrids ( •
• ) was made mid-way
of MMnCc hybrids (O
between divisions to give an average size for successive cell cycles after the transfer
operation. Since volume measurements cause some damage, one of each daughter
pair was used for measurement, then discarded; the other was allowed to grow and
divide undisturbed. The first point was obtained from measurement on hybrids before
division. These amoebae were not used for further measurements. Each point represents the average size of 20-50 amoebae.
536
M. J. Ord
put into the much smaller volume of Mini Mutant cytoplasm growth began immediately and by the time division took place cytoplasmic volume equalled that of
controls.
In SMnCc and PMnCc hybrids the change to mutant form was slow. In both types
of hybrids division was either retarded or halted immediately before the change to the
characteristic cytoplasmic crystal form and some time elapsed before a regular 5-day
cell cycle was resumed. No correlation was found between the number of divisions
after transfer and acquisition of mutant characteristics.
Table 3. The effect of contact of a control nucleus with Pale Mutant cytoplasm for limited
periods; that is, transfer of a control nucleus to Pale Mutant cytoplasm giving a CnPMc
hybrid, with removal of control nucleus after varying periods from 2-12 days and return
of Pale Mutant nucleus
Control nucleus
in mutant cytoplasm,
replaced by mutant
nucleus after
No. of transfers
2-3 days
11
State of
mutant cytoplasm Subsequent period
when control
during which mutant
nucleus was
characteristics were
removed
lost in the cytoplasm
Square platelet
crystals with
7-10 days
4- birefringence
6 days
8
7-8 days
14
12 days
10
Chiefly normal
isotropic crystals
with a few square
platelets
Chiefly normal
isotropic crystals
with a few square
platelets
Chiefly normal
isotropic crystals
with a few square
platelets
4 weeks
4—5 weeks
4-5 weeks
Some hybrids, slow to divide, assumed the mutant form after only 1-2 divisions;
others divided as many as 8-10 times before changing their crystal form. When cytoplasm was cut off at regular intervals to prevent division, mutant characteristics still
developed, proving that division was not necessary for the expression of mutant
characteristics. An attempt was made to speed up the change to the mutant form by
making multinucleate hybrids with 4 mutant nuclei each, cutting off cytoplasm to
prevent division. Even under such circumstances the change to mutant form still required 4 weeks. A second attempt was made to speed up the change to mutant form
by taking the unchanged offspring of 50 PMnCc hybrids and replacing their Pale
Mutant nuclei with fresh Pale Mutant nuclei 16, 23 and 28 days after the initial
transfer. Again the changeover time to mutant form was not significantly altered.
The rate at which cytoplasm lost its mutant characteristics when given a normal
Carcinogen-induced mutations in Amoeba
537
nucleus varied, but it was always faster than the change of normal cytoplasm by a
mutant nucleus. When normal nuclei replaced Pale or SpG Mutant nuclei normal
bipyramidal crystals generally appeared 3-4 days after the transfer operation, with a
change to 95 % normal crystals by 15 days. However, the complete loss of the last few
birefringent square platelets could take as long as 6 weeks and mass cultures from
CnSMt. hybrids sometimes retained a slight paleness even after several months.
In an attempt to find whether the control nucleus had any significant effect on
mutant cytoplasm when allowed to remain in it for only short periods, hybrids had
their control nucleus removed and replaced by a new mutant nucleus after varying
time intervals (Table 3). This experiment indicated that the presence of a control
nucleus could affect mutant cytoplasm sufficiently to produce a loss of mutant
characteristics after its removal even when no visible change had appeared during its
presence. No division of the control nucleus in the mutant cytoplasm was necessary
to bring about this change.
A study of anucleate Pale Mutant cytoplasm showed that the continued presence
of the Pale Mutant nucleus was not necessary for the existence of + birefringent
square platelets. Two hundred enucleated amoebae, followed until they cytolysed,
showed no loss of +birefringence. Starved Pale and SpG Mutant amoebae, in which
nuclear syntheses would be expected to be much reduced, showed no change to
normal crystals with starvation for 3-4 week periods.
The stability of SpG and Pale Mutant amoebae
Both SpG and Pale Mutant strains are unstable in that they will revert to normal
form. This entails not only the replacement of birefringent crystals by normal isotropic crystals, but also a return to normal size, cell cycle length, attachment and
feeding habits. In mass culture a small percentage of mutants continually change back
to normal, but provided normal forms are removed the rate of change is kept to less
than 1%. If normal forms are left in the culture, however, the change is far more
rapid. This is due both to the slow rate of division of the mutant form as compared
to the normal form and to the poor attachment of the mutant which allows considerable loss during each culture fluid change.
Although the reason for this reversal of form is not yet clear, two ways have been
found of increasing it: growing the mutants at higher temperature and in wheat
culture.
Increase in temperature. SpG Mutant amoebae were grown in an incubator at
26 ± 1 °C for a period of 9 months. Though some amoebae kept the SpG characteristics throughout the entire period many reverted to an 'apparent normal' form
(Fig. 2). This change was reversible. When these 'apparent normal' forms were returned to 18 °C the SpG Mutant characteristics gradually returned. Four groups of
SpG Mutant amoebae were put through a series of such changes (Table 4). Change to
'apparent normal' form at 26 °C took on average 30 days; change from 'apparent
normal' to mutant form with a return to 18 CC took on average 26 days. Growing
the 'apparent normal' forms at 15 °C instead of 18 °C decreased the time needed for
SpG
I
II
Pale
Mutants
I
II
III
IV
Mutants
3°
33
17
5°
60
55
25
28
1 /
22
21
70
75
17
15
22
I /
24
38
38
I /
I /
32
45
34
28
45
15
19
40
15
34
24
35
I /
27
35
25
I /
24
'Apparent
' Apparent
'Apparent
Mutant form ; normal' form;
Mutant form;
normal' form;
Mutant form ;
normal' form;
changed by
changed by
changed by
changed by
changed by
changed by
growing at
growing at
growing at
growing at
growing at
growing at
26 °C for
18 °C for
26 °C for
18 °C for
26 °C for
i8°Cfor
no. of days
/ no. of days
/ no. of days
/ no. of days
/ no. of days
/ no. of days
f
stated
to
stated
to
stated
to
stated
to
stated
to
stated
to
Mutant form
(Each group represents initially 25 amoebae. Though divisions increased the numbers in each dish, only 25 amoebae were carried on at each change
to clean dishes. Changing was carried out using dark field illumination to prevent any selection of paler or darker forms.)
Table 4. Changing SpG and Pale Mutant amoebae back and forth between mutant and an 'apparent normal' form
by raising and lowering the temperature from 18 °C {giving mutant form) to 26 °C {giving the 'apparent normal' form)
Carcinogen-induced mutations in Amoeba
539
a return to SpG Mutant form by about 30%. Normal A. proteus did not at any time
change their character at temperatures from 15-28 °C.
Wheat culture. SpG Mutant amoebae put into wheat mass cultures began forming
normal isotropic bipyramidal crystals within 5 days; 60% of the amoebae assumed an
'apparent normal' form by 14 days. No SpG Mutant amoebae with +birefringence
remained after 2 months, though paler-than-normal amoebae could always be found
throughout the 9-month period. This change was also reversible. Groups of these
26 °c
26 °C
18 °C
All mutant
form
(+birefringence)
All 'apparent
normal' form
(—birefringence)
18 °C
r\
, , AJ, , A, ,/ , ,v, J.
20
40
60
80
100 120
Days
140
160
180
200
220
Fig. 2. The change to 'apparent normal' form of SpG Mutants produced by a rise in
temperature to 26 °C and reversed by a decrease in temperature to 18 °C. In the
figure, one clone has been followed through an 8-month period during which time
amoebae were moved back and forth 7 times between 26 and 18 °C as they changed
to 'apparent normal' form and back to mutant form.
Table 5. Return of mutant characteristics in SpG and Pale Mutant amoebae
after a temporary change to normal form by growing in wheat culture
Type of
mutant
Pale Mutant
amoebae which
had assumed
normal form in
wheat culture
SpG Mutant
amoebae which
had assumed
normal form in
wheat culture
Period
spent
in wheat
culture,
weeks
Time required
to return to
mutant
form
once removed
from wheat,
months
IS
3-5
6
6
4'5
4
4
2
2-5
3
2
6
9
2
2
3
6
9
12
12
IS
i-5
i'S
i-5
54 o
M.J.Ord
'apparent normal' forms removed from wheat after 2, 3, 6, 9, 12 and 15 weeks returned to the SpG Mutant form, though they frequently took over 2 months to do
so (Table 5). The longer period in wheat did not increase the time required for this
change.
Pale Mutant amoebae underwent the same changes with both temperature and
wheat as did the SpG Mutant amoebae (Tables 4 and 5), but with this mutant the
average time to change from the 'apparent normal' form back to the Pale Mutant
form was much longer.
Table 6. Heterokaryons composed of mutant and control nuclei in
either mutant or control cytoplasm
(Heterologous nuclei remained in close contact for from 2-6 weeks. Change in the
form of the cytoplasmic crystals was used as an indication of the expression of the
mutant or control nucleus.)
Type of
heterokaryon
No.
Period of
contact
between
heterologous
nuclei,
weeks
Square platelet
crystals, mutant
type; crystals
with + birefringence
Bipyramidal
crystals,
control type;
isotropic
10% developed a
No loss of normal
small number of
bipyramidal crystals
mutant-type square
platelets after approx.
5 weeks.
11
Those surviving for
No loss of
2-4
3PMnCnCc*
over 3 weeks
bipyramidal
developed a small
crystals
number of mutanttype square platelets
with + birefringence
Most square platelets
CnPMnPMc
26
Bipyramidal crystals
2-4
developed within
and + birefringence
4-5 days and were
disappeared in 1-2
weeks; all mutant-type
numerous after
crystals disappeared
1 week
from cytoplasm by
3 weeks
Square platelets with
CnSMnSMc
Bipyramidal crystals
3-5
17
+ birefringence
appeared in most
disappeared by 3 weeks
amoebae and were
in all but 3 amoebae;
numerous in 70%
these retained some
of the heterokaryons
square platelets
by 2-3 weeks
throughout the
experiment
* Addition of 3 mutant nuclei with one control nucleus.
PMnCnCc
5°
4-6
541
Carcinogen-induced mutations in Amoeba
Interaction between mutant and control nuclei sharing the same cytoplasm
The effect of nuclear syntheses of one type of nucleus upon the other was observed
in heterokaryons during periods of 2-6 weeks by cutting off pieces of cytoplasm at
regular intervals to prevent division while allowing growth (Table 6). When control
cytoplasm acted as the container for the heterologous nuclei, there was no loss of
normal bipyramidal crystals at any time, and few amoebae showed mutant characteristics. When, on the other hand, mutant cytoplasm acted as the container for
Table 7. Results of 3 experiments in which heterokaryons were allowed to divide after
heterologous nuclei had shared the same cytoplasm for from 4 days to 5 weeks
(Offspring of the division were cloned singly to observe the effect close contact between the heterologous nuclei had on subsequent nuclear expression. Division of
heterokaryons may be into 4 mononucleate daughters; 1 binucleate and 2 mononucleate daughters; 2 binucleate daughters; 1 trinucleate and 1 mononucleate
daughter. Since the carrying of a mutant nucleus in a binucleate with one normal
nucleus masks the expression of mutant characteristics, in expts. 2 and 3 all daughters
of heterokaryons with more than one nucleus had their nuclei transferred to control
cytoplasm. Since mutant amoebae had a much lower viability than control amoebae,
non-viable offspring would in most cases be amoebae carrying the mutant nucleus.
In some cases this was apparent by the appearance of square platelet crystals, but not
all amoebae survived long enough to show such changes.)
Expt.
no.
1
2
3
Type of
heterokaryon
CnSMnSM,:
CnPMnPM,
CnSM1tSM,
Period of
contact between
heterologous
nuclei before
division was
permitted,
No.
weeks
16
5
'4
No. where £
offspring
produced
normal and
$ mutant
clones
4-6
4
7
5
2-5
11
No. where £
offspring produced
No. producing
normal clones
normal clones
and £ non-viable
clones, frequently
only but with
a high % of
with mutant
binucleate amoebae
characteristics
5
—
3
4
—
•—
heterologous nuclei, normal crystals appeared in the mutant cytoplasm by one week
and square platelets gradually disappeared. Disappearance of the mutant-type crystal
was slower in the presence of the mutant nucleus than in its absence, as shown by
comparison with hybrids containing control nuclei in mutant cytoplasm. In no
instance was the expression of the characteristics of the control nucleus suppressed
or masked. In heterokaryons containing the control and SpG Mutant nuclei in
mutant cytoplasm, mutant characteristics were retained, along with the developing
normal characteristics, for much longer periods than in heterokaryons with a control
and a Pale Mutant nucleus in Pale Mutant cytoplasm. This suggests that the SpG
Mutant nucleus was less easily influenced by the control nucleus.
The effect on heterologous nuclei of sharing the same cytoplasm during division
was observed also. At this time nuclear protein, RNA and nucleoli move out of the
nuclear envelope. Heterokaryons allowed to divide after contact between nuclei of
542
M. J. Ord
from 4 days to 5 weeks gave rise to daughter cells which produced both mutant and
normal clones (Table 7). In a few cases only normal clones were obtained. In these,
division produced either non-viable daughter cells or binucleate or trinucleate daughters where expression of mutant characteristics could have been masked by the
presence of a control nucleus. The possible masking of a mutant nucleus in this way
was prevented in later experiments by transplanting the nuclei of all binucleate or
multinucleate amoebae to fresh control cytoplasm.
DISCUSSION
The transfer of nuclei between normal and mutant amoebae shows, without exception, that the mutant characters, even those expressed in the cytoplasm, are due to
a change in the nucleus. Experiments with heterokaryons indicate that, in all probability, it is the chromatin of the nucleus which has been affected. This deduction is
possible because at prophase the nucleoli, nuclear protein and nuclear RNA of an
amoeba leave the nucleus and disperse in the cytoplasm (Prescott & Goldstein, 1968).
In a heterokaryon these nuclear constituents would intermingle in a common cytoplasm. The chromatin of the nucleus, which stays within the nuclear membrane, remains separate since even at metaphase this envelope only partially breaks down. No
fusion of nuclei or sharing of a metaphase plate takes place when 2 or more nuclei
divide in a common cytoplasm. If the 'mutant change' was carried by a nuclear constituent, which in heterokaryons mixed with that of a normal nucleus at division,
some mixture of mutant and normal characteristics would be expected. No mixing
occurred. Offspring produced by the division of heterokaryons contained either a
mutant nucleus or a normal nucleus and with few exceptions each heterokaryon gave
rise to both mutant and normal clones.
The lack of clones with intermediate characteristics and the lack of an antagonistic
action between mutant and normal nuclei proves that mutants produced by MNU
treatment are unlike the naturally occurring or selected strains used by other workers
(Yudin & Nikolajeva, 1968; Lorch & Jeon, 1969). Poor survival of the offspring of
heterokaryons between such strains is common, and in many cases antagonistic action
between nuclei is such that almost all are lethally damaged (Jeon, 1969). Makhlin &
Yudin (1969), studying this antagonistic action between heterologous nuclei in
amoebae heterokaryons, showed that when a donor nucleus was introduced into a
heterologous amoeba minute quantities of a 'specific substance' sufficient to destabilize or lethally damage the host nucleus moved from the donor nucleus in less than
a minute.
Jeon & Lorch (1969), in heterokaryons involving a number of strains of A. proteus,
found the nucleus to be the site of origin and of action of a ' lethal factor' which remained stable even outside the cell but was not connected with either RNA or DNA.
Goldstein & Prescott (1967), studying amoeba proteins, have shown that a type of
nuclear protein exists which moves rapidly from one nucleus to another in heterokaryons. Makhlin & Yudin's 'specific substance' or Jeon & Lorch's 'lethal factor'
could form a fraction of such a protein. In the experiment reported here the lack of
Carcinogen-induced mutations in Amoeba
543
any antagonism between heterologous nuclei indicates that, if the Xdy proteus strain
of amoeba used for this study contains a specific substance or lethal factor, it has
probably not been altered by the mutagen since mutant and control nuclei and cytoplasms are still compatible. This was well shown in heterokaryons where division
was prevented by cutting off pieces of cytoplasm for periods of 4 or more weeks.
Despite the very long period of contact, the heterologous nuclei were neither damaged
nor changed and once separated gave rise to clones of either mutant or control
amoebae.
During the period in which mutant and control nuclei shared the same cytoplasm,
it was the control nucleus that determined the character of the heterokaryon. If the
container for heterologous nuclei was normal cytoplasm, few if any of the mutant form
of cytoplasmic crystals appeared. If, on the other hand, mutant cytoplasm acted as the
container for control and mutant nuclei, normal crystals appeared within a week and
rapidly increased in number, while mutant crystals decreased. By the end of 3 weeks
most heterokaryons lost all mutant characteristics and only separation of the nuclei
allowed the expression of mutant characteristics again.
TV-methyl-A'-nitrosourethane is one of a group of nitroso compounds, many of
which are both potent carcinogens and mutagens (Magee & Barnes, 1967). The way
in which they produce mutations is as yet unknown, though it has been shown that
nitroso compounds are able to methylate DNA, RNA and protein (Magee & Faber,
1962; Magee & Hultin, 1962), and that the guanidine base of RNA and DNA is
particularly vulnerable to such methylation in the 7 N position (Craddock & Magee,
1963). Nitrosamides, such as A?-methyl-A?-nitrosourethane and A^-methyl-A^-nitrosoAf'-nitroguanidine, lend themselves particularly well to studies of mutagenesis since:
(1) they break down to active derivatives rapidly and spontaneously in aqueous
solution, with a particular affinity for SH groups (Schoental & Rive, 1965); (2) the
active derivatives have a very short life; and (3) the period of exposure necessary to
produce changes in single cells, e.g. amoebae, is very short in comparison with the
length of the cell cycle (Ord, 1968 a). The shortness of the exposure time with such
substances means that the activities occurring within the cell, or the condition of the
cell at the time of exposure to the mutagen, determine to a large extent the magnitude
and type of damage to the cell.
Despite the known potency of MNU in producing mutations, changes of this type
were not expected in amoebae. The nucleus of A. proteus has long been considered
polyploid, though the existence of polyploidy is based on circumstantial evidence.
For example, the very large number of chromosomes (chromosome number for the
X6y and related strains is approximately 1000 (L. G. E. Bell, unpublished)); the
large number of nucleoli; the multinucleate form taken by other large free-living
freshwater amoebae; the distribution of labelled DNA through successive divisions
(Ord, 19686); the ineffectiveness of mutagens such as bromouridine (M. J. Ord, unpublished); the resistance of amoebae to X-rays (Ord & Danielli, 1956); the similarity
in LD 50 X-ray dose for Pelomyxa carolinensis, with several hundred small nuclei,
(Daniels, 1956) and A. proteus, with a single large nucleus. A mutation in a cell
which has many representatives of each gene would require a change in all, or at
544
M.J.Ord
least many, of these. Where the mutagen is present for only short periods it would be
necessary for all copies of the gene to be vulnerable at the same time, while the
majority of other genes were protected. These requirements are unnecessary in a
diploid cell where misreading of a single copy of a gene leads to a mutation. Two
activities of chromatin would be expected to leave genes vulnerable to a mutagen:
DNA replication and DNA transcription. Providing that during DNA replication all
equivalent loci replicate their DNA at the same time, or that during transcription all
copies of a given gene 'switch on' simultaneously, these periods would give vulnerability to particular genes while other periods would give less. If, for example, mutations
occurred due to alteration of the DNA by the mutagen during replication, 'changed
strains' would be expected only from amoebae in S at the time of treatment. If, on
the other hand, mutations occurred due to alteration of the DNA during transcription, ' changed strains' would be expected from amoebae treated at times other than S.
Experiments where amoebae are treated with the mutagen every 2 h throughout the
48-h cell cycle prove that mutations, though occasionally arising during S, are generally produced at other times in the cell cycle (M. J. Ord, unpublished). This suggests
the involvement of DNA transcription. A study of RNA and protein syntheses
during the cell cycle shows a correlation between these periods and those when mutations most frequently arise. Further experiments are in progress investigating the
part played by the functional state of the DNA in the mutagenic process.
The author is indebted to Mr R. Legg for assistance with photography.
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M. J. Ord
Fig. 3. Pale Mutant and parent strain X67 amoebae photographed through crossed
Polaroid filters with half extinction. This shows the + birefringence of the Pale Mutant cytoplasmic crystals. Parent amoebae with isotropic crystals appear dark, x 120.
Fig. 4. Cytoplasm of Pale Mutant and parent strain X67 amoebae. The control cytoplasm (left) has truncated bipyramidal crystals of 1-2 /tm long diameter. The Pale
Mutant cytoplasm (right) has square or rectangular platelets averaging 5-10 /tm along
the edge and appearing needle-shaped when viewed on edge, x 550.
Fig. 5. Crystals of the Pale Mutant cytoplasm in a squashed amoeba to show the flat
square or rectangular shape, x 600.
Carcinogen-induced mutations in Amoeba
..-.*»
M. J. Ord
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Fig. 6. Mini Mutant amoeba (left), amoeba of parent strain X67 (right), x 250.
Fig. 7. SpG Mutant and parent strain X6y amoeba. Note the ghost-like appearance
of the mutant which is moving above and very close to the control amoeba, x 200.