y. Cell Sd. 72, 111-120 (1984)
111
Printed in Great Britain © The Company of Biologists Limited 1984
DESTRUCTION OF IMMATURIN ACTIVITY IN
EARLY MATURE MUTANTS OF PARAMECIUM
CAUDATUM
ISOJI MIWA
Biological laboratory, College of General Education, Ibaraki University,
Mito310,jfapan
SUMMARY
Cells of Paramecium in the sexually immature period of the clonal life cycle contain a protein
called immaturin, which represses mating activity when injected into sexually mature cells. The
amount of immaturin at various stages of the immaturity period was examined by injecting
cytoplasm from immature cells of one wild-type and two early mature mutant clones into mature
cells of wild-type. The proportion of immature cells brought about by the injection increased until
about 10-15 fissions after conjugation of the donor cells in all three clones. But the percentage of
immature cells induced by the injection decreased rapidly from the 25th to the 30th fission of both
early mature donors, while the percentage was still high with a wild-type donor of the same fission
age. When cytoplasm of the Emt A (early mature mutant) homozygote taken immediately before
maturation (about 27 fissions) was injected into the wild-type immature cells of 45 fissions after
conjugation, 13 % of the recipient cells expressed mating reactivity. But when cytoplasm of Emt A
cells 20 and 50 fissions after conjugation was injected into the same cells, no reactive cell appeared.
The results suggest that cytoplasm of the early mature mutant immediately before maturation
contains some material that accelerates maturation when injected into wild-type cells in the late stage
of immaturity. On the other hand, the immature cytoplasm of wild-type cells about 25 fissions after
conjugation showed no effect when injected into early mature cells immediately before maturation.
The function of the early mature gene product was suggested to be the destruction of immaturin
activity.
INTRODUCTION
An exconjugant clone of Paramecium has a period of immaturity, during which
time the animals cannot mate even when they enter stationary phase. The length of
immaturity depends upon the number of fissions after conjugation (Sonneborn, 1957;
Miwa & Hiwatashi, 1970, and others). As already reported, cells of Paramecium
caudatum in the immaturity period contain a substance called immaturin, which
represses the expression of mating activity when injected into sexually mature cells
(Miwa, Haga & Hiwatashi, 1975). Immaturin has been isolated and characterized. It
is a heat-labile protein with a molecular weight of about 10000 (Haga & Hiwatashi,
1981) and is common to three different species complexes in Paramecium (Miwa,
1979a,6). The question is how does immaturin control the length of immaturity.
Myohara & Hiwatashi (1978) reported two early mature mutants with incomplete
dominance in P. caudatum. This suggests that the duration of the immaturity period
Key words: Paramecium caudatum, immaturin, early mature mutants.
112
I.Miwa
in this species is controlled genetically, as in other ciliates (Siegel, 1961; Bleyman &
Simon, 1967). There are various possible reasons why the early mature mutants
mature early: (1) the amount of immaturin may decrease rapidly or precociously in
the mutants. (2) The amount of immaturin and its rise and fall may be normal, but
the receptor for immaturin may become defective precociously. (3) The early maturity might be brought about by a mechanism completely different from the functions
of immaturin and its receptor.
In the present attempt to discover the function of the early mature genes, the rise
and fall in the amount of immaturin during the course of the immaturity period in
clones of wild-type and early mature mutants were first observed, and then the action
of the cytoplasm of the early mature mutants immediately before they reached maturity was examined. The results of these experiments suggest a possible mode of action
of the early mature gene.
MATERIALS AND METHODS
All strains used belong to P. candatum, syngen 3. The wild-type strains were dm-21 (mating type
V) and dm-23 (mating type VI), both of which were F3 progeny from a cross between natural stocks
Kokl and Koj. The strains of early mature mutant were 27aG3 (mating type V) and Cys-9 (mating
type VI) both of which were homozygous for the early mature gene Emt A, and also EmIII-3s
(mating type VI), a homozygote of the early mature gene Emt B. All early mature strains were
supplied by K. Myohara. Immature cells used as donors for the microinjection of cytoplasm were
progeny from a cross between dm-21 and dm-23 of wild-type, from a cross between 27aG3 and Cys9 of the Emt A homozygote and from a selfing pair of EmI II-3s of the Emt B homozygote. The stocks
used as testers for mating reactivity were 10A212 (mating type V) and 16A101 (mating type VI) of
the CNR mutant (no backward swimming, Takahashi, 1979).
All stocks and strains in the present experiment were cultured in fresh lettuce medium inoculated
with Klebsiella pneumoniae one day before use (Hiwatashi, 1968). Cultures were kept at 25 °C. The
number of fissions after conjugation was calculated by use of the daily isolation method (Miwa,
1973).
Microinjection of cytoplasm was performed with a micromanipulator of de Fonbrune type
(Narishige, Tokyo) with a single needle, according to Koizumi (1974). Recipient cells after injection
were kept for 3 h in the cell-free culture supernatant from the stationary-phase culture of the
recipient and then grown in a microcapillary tube (Drummond) containing 2 n 1 of culture medium.
Mating reactivity was observed in depression slides by adding about 100 highly reactive living cells
of the complementary mating type. When the recipient cells were in the immature period, the test
of mating reactivity was performed twice with CNR mutants of mating type V and VI. After the test,
the recipient cells were picked up by observing backward swimming in the test solution containing
25 mM-KCl in Dryl's solution (Dryl, 1959) in which CNR cells never showed backward swimming.
After the first mating reactivity test with the tester of one mating type of the CNR mutant, recipient
cells were isolated into adaptation medium (modified Dryl's solution with KH2PO4) and then tested
for mating reactivity with the tester of the other mating type.
RESULTS
Quantitative changes in immaturin during the immaturity period of ivild-type clones
About 10 pi of cytoplasm from immature cells at various times after conjugation was
injected into mating-reactive cells of stock dm-23. In the early three stages of the
immature period, 30 different injections were made at each stage and then at later
stages, 25 different injections with five donor cells each from five immature clones
Destruction ofimmaturin in P. caudatum
113
were made at each stage. As a control, the same volume of mature cytoplasm from
mating-reactive cells was injected into the cells of the same stock. After injection each
recipient cell was kept for 3 h in the cell-free supernatant from a stationary-phase
culture, and then grown in a microcapillary containing 2/x] of fresh culture medium.
Each injected cell divided twice until 2 days after injection. Mating reactivity of the
recipient clones was tested 2 days after injection by adding about 100 cells of the
complementary mating type. Fig. 1 shows the percentage of non-reactive cells in
recipient clones from injected cells. The ratio of immature cells brought about by
injection increased until about 10-15fissionsafter conjugation of donor cells and then
decreased gradually. In the control, which was injected with mature cytoplasm, the
percentage of non-reactive cells was always less than 10%. Though the ordinate of
Fig. 1 is the percentage of effective injections, the curve suggests a rise and fall in the
amount of immaturin in donor cells during the course of the immaturity period. The
next experiment was aimed at determining an equivalent curve in early mature
mutants.
Absence of an effective amount ofimmaturin in early mature mutant cells immediately after maturation
Two early mature genes, Emt A and Emt B, are incompletely dominant and their
homozygotes and heterozygotes have immaturity periods of 25 and 15fissionsshorter,
respectively, than that of wild-type (about 55fissions)(Myohara & Hiwatashi, 1978).
To know whether the shortened immaturity period is due either to precocious
decrease in immaturin or to inactivation of the supposed immaturin receptor, the
100
(0
ID
50
Mature
O
10
20
30
40
Fissions after conjugation
50
Fig. 1. Quantitative change in immaturin during the immaturity period of wild-type
clones. About 10 pi of cytoplasm from immature cells at various times after conjugation
was injected into mating-reacting cells of stock d m -23. After injection each recipient cell
was kept for 3 h in the cell-free supernatant from stationary-phase culture, and then grown
in a microcapillary containing 2/zl of fresh culture medium. Each injected cell divided
twice until 2 days after injection. Mating reactivity of the recipient clones was tested 2 days
after injection by adding about 100 cells of the complementary mating type. Bars indicate
95 % confidence ranges.
60
114
/. Miwa
Table 1. No effect of the cytoplasm of the early mature mutant 30 fissions after
conjugation was found when it was injected into mating reactive wild-type cells
Frequency of recipient clones that show various ratios of
mating-reactive cells*
Donor
Early mature mutant cellsf
Wild-type cellsj
0/4
1/4
2/4
3/4
4/4
Total
0
0
0
1
2
1
9
6
14
17
25
25
• Each injected cell was grown for 2 days in a microcapillary. Mating reactivity in recipient clones
(stock dm-23) two fissions after injection was tested with about 100 highly reactive cells of tester
(dm-21).
| Progeny of a cross between stocks 27aG3 and Cys-9, immediately after maturation.
X Mating-reactive cells of stock d m -23.
amount of immaturin in early mature cells immediately after maturation was
examined. About 10 pi cytoplasm of the early mature Emt A homozygotes about 30
fissions after conjugation was injected into mating-reactive wild-type cells. The
mating reactivity of the recipient clones was tested two fissions after injection by
adding tester cells. As seen in Table 1, the mutant contained no effective immaturin
at this stage. Since the result suggests a precocious decrease in immaturin in the early
mature mutant, the next experiment was done to examine the changes in the amount
of immaturin in exconjugant clones of early mature mutants.
Quantitative changes in immaturin during the immaturity of early mature mutants
About 10 pi of immature cytoplasm of an Emt A homozygote, produced by a cross
between strains 27aG3 and Cys-9, was injected into mating-reactive cells of wild-type.
Injections were made at various stages from immediate exconjugants to mature cultures. At each stage of injection, 25 donor cells,fivecells each from five immature Emt
A clones, were used. About 3 h after injection, each recipient cell was transferred into
2^1 of fresh culture medium in a microcapillary and grown for 2 days, during which
period most cells divided twice. The mating reactivity of recipient clones was tested
as before. The percentage of non-reactive cells rose from the exconjugant stage to 10
fissions after conjugation of the donor. This rise of the curve is the same as when wildtype exconjugant clones were used as the donor. But the percentage fell rapidly by
25—30 fissions (Fig. 2). A very similar experiment was done using a homozygote of
.the other early mature mutant, Emt B, as the donor. Immature clones homozygous
for Emt B were obtained from self-conjugation of stock EmIII-3s. The pattern of rise
and fall of the percentage of non-reactive cells by the injection of immature Emt B
cytoplasm was similar to that observed with the Emt A homozygote (Fig. 3).
Injection of cytoplasm from early mature mutants immediately before maturation
As seen in Figs 2 and 3, a rapid fall in immaturin content was observed around 25
fissions in the early mature mutants. This fall seems to suggest that active destruction
Destruction of immaturin in P. caudatum
100
115
Emt A/Emt A
0)
I 50
o
Mature
z
10
20
30
40
50
Fissions after conjugation
Fig. 2. Quantitative change in immaturin during the immaturity period of early mature
mutants. Immature cytoplasm of an Emt A homozygote was injected into mating-reactive
cells of wild-type. The conditions of injection and test of mating reactivity were the same
as for Fig. 1.
100
Emt B/Emt B
5
o
CD
I
?
c
o
Z
Mature
10
20
30
Fissions after conjugation
40
50
Fig. 3. Quantitative change in immaturin during the immaturity period of the early
mature mutant clones, Emt B homozygote. The conditions of injection and test of mating
reactivity were the same as for Fig. 1.
of immaturin occurs in the early mature mutants immediately before maturation. The
following experiments were performed to test the above possibility. In the first experiment, cells of Emt A homozygote about 27 fissions after conjugation were used as
donors of cytoplasm. Recipients were wild-type cells of three different stages:
immature cells of about 25 and 45 fissions, and mature cells of about 65 fissions after
conjugation (Fig. 4). The conditions of injection, treatment of recipient cells and
/. Miwa
116
mating-reactivity test were the same as before. The results are shown in Table 2.
When the recipients were immature cells of 25 fissions after conjugation (A) no mating
reactivity appeared after injection. But when the recipients were immature cells of 45
fissions after conjugation (B), 13 % of the injected cells expressed mating reactivity
two fissions after injection, while non-injected cells of the same clone expressed no
mating reactivity until about 55 fissions after conjugation. When recipient cells of 65
fissions after conjugation (C) were injected, they showed normal mating reactivity. As
controls for the above experiment, cytoplasm oiEmtA homozygote cells of 20 and 50
fissions age and cytoplasm of wild-type cells of 55 fission age (immediately before
maturation) were injected into the 45 fission cells of the wild-type. No mating-reactive
cell appeared in these injections. The results suggest that the cytoplasm of the early
mature mutant immediately before maturation contains some substance(s) that accelerates maturation when injected into wild-type cells in the late stage of immaturity.
If the action of this substance(s) is to destroy the function of immaturin, the injection
Immaturity
Maturity
27
Early mature
mutant
{EmtA/EmtA)
o
Wild-type
25
65
Fissions
Fig. 4. Injection of cytoplasm from early mature mutant immediately before maturation
into wild-type cells. The cytoplasm of the early mature mutant, Emt A homozygote, about
27 fissions after conjugation was injected into wild-type cells at three different stages,
immature stages of about 25 and 45 fissions, and mature stage of about 65 fissions after
conjugation.
Table 2. Expression of mating reactivity in wild-type cells of late immature stage by
injection with cytoplasm from the early mature mutant immediately before maturation
Injection*
A
B
C
No. of clones in
recipient
Mating-reactive cells
two fissions after
injection')'
Mating-reactive cells in
non-injected
recipients!
25
25
25
0/100
13/100
82/100
0/100
0/100
87/100
•As in Fig. 4.
f Each recipient cell was grown for 2 days in a microcapillary. Mating reactivity in recipient clones
was tested twice with CNR mutants of mating types V and VI when the recipient cells were in the
immature period, and with a tester of complementary mating type when the recipients were in the
mature period.
Destruction of immaturin in P. caudatum
117
of immature cytoplasm into the early mature cells immediately before maturation
would be ineffective. The next experiment was done to test this hypothesis.
Injection of immature cytoplasm from wild-type into the early mature mutant immediately before maturation
The immature cytoplasm of wild-type cells of about 25 fissions was injected into
early mature cells (Emt A homozygote) immediately before maturation (27 fissions).
As controls, the same cytoplasm was injected into early mature mutant cells of 20 and
55 fissions and into wild-type cells immediately before maturation (55 fissions) (Fig.
5). The conditions of injection and the tests for mating reactivity were the same as
before. The results are shown in Table 3. The recipient cells of injection A, that is
the early mature cells of 20 fissions, showed no mating reactivity. The recipients of
injection B, which are the early mature cells immediately before maturation (27
fissions), showed 65 % mating reactivity. The recipients of injections C and D showed
only about 20 % mating reactivity. The most interesting difference is seen between
Early mature 0
mutant
(Emt A/Emt A)
Immaturity
55
Maturity
Wild-type
Fissions
55
Fig. 5. Injection of immature cytoplasm from wild-type into the early mature mutant.
The immature cytoplasm of wild-type cells about 25 fissions was injected into the early
mature cells (Emt A homozygote) at three different stages, immaturity, immediately
before maturation and maturity, and into wild-type cells immediately before maturation.
Table 3. No effect of immaturin in immature cells of wild-type was found when it was
injected into the early mature mutant immediately before maturation
Injection*
A
B
C
D
No. of clones in
recipient
Mating-reactive cells
two fissions after
injectionf
Mating-reactive cells in
non-injected
recipients!
25
25
25
25
0/100
65/100
22/100
19/100
0/100
63/100
90/100
58/100
•As in Fig. 5.
f Each recipient cell was grown for 2 days in a microcapillary. Mating reactivity in recipient clones
was tested twice with CNR mutants of mating type V and VI in injection A, B and D, and with a
tester of complementary mating type in injection C.
118
I.Miwa
injections B (early mature recipient) and D (wild-type recipient) when both recipients
are in the stage immediately before maturation. These results again suggest that cells
of the early mature mutant in the stage immediately before maturation contain a
mechanism that inactivates the immaturin.
DISCUSSION
In this work, a direct measurement of the protein, immaturin, has not been made
but the relative amount of immaturin contained in the cytoplasm was estimated from
the percentage of immature cells produced by the injection of immature cytoplasm.
Since it is known that when purified immaturin is injected into mature cells, the
percentage of immature cells brought about by the injection is proportional to the dose
of immaturin injected (Haga, 1979), the number of immature cells obtained by
injecting immature cytoplasm should reflect the amount of immaturin contained in
the donor cells. Thus, the rise and fall in the percentage of non-reactive cells shown
in Figs 1, 2 and 3 reflect changes in the relative amounts of immaturin contained in
cells at various stages during the immature period.
The amount of immaturin present immediately after conjugation is small in the
wild-type as well as in early mature mutants. Nevertheless, most exconjugant clones
show immaturity even in the very early period after conjugation. Probably, the total
amount of immaturin in a cell at this stage is barely sufficient to inhibit mating
reactivity. Since the volume of cytoplasm transferred by injection is about 1/30 of the
whole cell volume, the percentage of non-reactive cells brought about by the injection
of cytoplasm at this stage would be low. This interpretation is supported by the fact
reported by Hiwatashi (1958) that some exconjugant clones show mating reactivity for
a short time immediately after conjugation and then become immature. In the present
experiments also, cells of some exconjugant clones derived from the cross between
dm-21 and dm-23 showed mating reactivity for less than 10 fissions after conjugation
and then became immature.
A conspicuous feature seen in early mature mutants is the precocious and abrupt
fall in the curve by 25-30 fissions after conjugation (Figs 2, 3), which has never been
observed in wild-type clones. This drop suggests that there is either an interruption
of the synthesis of immaturin or an inactivation of immaturin already synthesized. If
synthesis were interrupted, the fall in the curve should be more gradual, because when
immaturin is injected into mature cells, the immaturity brought about by the injection
continues for as long as 20 fissions (Haga, 1979). The possibility of the inactivation
of immaturin is supported by the two different injection experiments shown schematically in Figs 4 and 5. In the first experiment, where cytoplasm of the early mature
mutant immediately before maturation (27 fissions) was injected into wild-type cells
at three different stages, some recipient cells in the late immaturity period became
mature, while other recipients showed no difference from non-injected controls. In
the second experiment, where cytoplasm of wild-type cells in the stage containing an
almost maximal amount of immaturin (25 fissions) was injected into early mature
mutants at three different stages and wild-type cells at the beginning of maturity, the
Destruction of immaturin in P. caudatum
119
early mature recipient immediately before maturation (27 fissions) showed high reactivity, while the early mature recipient at complete maturity and the wild-type
recipient at the beginning of maturity became immature at high frequency. Since the
early mature recipients of 27 fissions were tested for mating reactivity two fissions after
injection, the stage of the recipients tested was near the beginning of maturation and,
thus, they should exhibit high mating reactivity. These experiments suggest that
cytoplasm of the early mature mutants immediately before maturation destroys
immaturity of wild-type cells in the late stage of the immaturity period, and that
immaturin from wild-type cells is destroyed in the early mature cells immediately
before maturation. These results lead to the conclusion that early mature mutant cells
in the stage immediately before maturation contain a factor that inactivates
immaturin. The injection of cytoplasm from the early mature mutant at 27 fissions
into wild-type cells of 25 fissions was ineffective. This result indicates that the amount
of immaturin contained in the cells of 25 fissions was too large to be destroyed by the
small amount of injected inactivating factor. Whether the inactivating factor found in
the early mature mutant is a direct product of the early mature gene remains uncertain. What mechanisms regulate the rise and fall of the amount of immaturin in
wild-type cells also remain to be determined.
This paper is dedicated to the memory of the late Dr Koji Myohara. The author thanks Dr K.
Hiwatashi for help in preparation of the manuscript.
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