Jpn. J. Protozool. Vol. 49, No. 1, 2. (2016)27

Regular Article

Rapid response to nutrient depletion on theexpression of mating pheromone, gamone 1,
in Blepharisma japonicum
Mayumi SUGIURA1, Mika YAMANAKA1, Toshinobu SUZAKI2 andTerue HARUMOTO1*
1

Department of Chemistry, Biology, and Environmental Sciences, Faculty of Science, Nara
Women’s University, Nara 630-8506, Japan2
Department of Biology, Graduate School of Science, Kobe University, Kobe 657-8501,
Japan

SUMMARY
Ciliate conjugation usually occurs when cells are moderately starved after rapid proliferation. In
Blepharisma japonicum, at the stationary phase, a mating pheromone (gamone 1) is secreted by matingtype I cells and stimulates mating type II cells to secrete another mating pheromone (gamone 2), which
stimulates mating type I cells. Both stimulated cells are transformed to be able to form pairs. We showed
previously that the gamone 1 transcription occurs only in the stationary-phase cells, not in logarithmicphase cells. However, it is still unknown how rapidly cells response to starvation and express gamone 1.
Here we show that gamone 1 expression is initiated by sudden artificial starvation, and we determined
the time required for the initiation of transcription and secretion of gamone 1 in B. japonicum.
Proliferating cells rapidly responded to artificial starvation, approx. 1.5 h at most was necessary for the
initiation of the transcription of gamone 1 gene, and approx. 3 h was needed for the secretion of gamone
1. Gamone 1 expression tested in single cells suggests that gamone 1 expression may be correlated withthe cell-cycle, most probably with the certain stage of G1 phase in B. japonicum. Our present findings
provide an experimental system to investigate the environmental factors involved in the initiation of
conjugation.
Key words: Conjugation, Starvation, Cell cycle*Corresponding author
Tel/Fax: +81-742-20-3421
E-mail: harumoto@cc.nara-wu.ac.jp
Received: 22 March 2016; Accepted: 18 April 2016.

28

Rapid response of gamone 1 expression by starvation

INTRODUCTION
Conjugation in ciliates, a process of sexual
reproduction which generates new progenies, usually occurs when cells are moderately starved after
rapid proliferation. Cells start interacting between
complementary mating-type cells and initiate conjugation. In the case of Blepharisma japonicum, amating pheromone (gamone 1), which is an approx. 30-kDa glycoprotein, is initially secreted bymating type I cells (Miyake, 1981a, 1981b, 1996,
for review). The secretion of gamone 1 transforms
mating type II cells to enable them to form conjugating pairs. Stimulated mating type II cells also
start secreting another mating pheromone (gamone2), which is a tryptophan-derivative compound
resembling serotonin. The secretion of gamone 2transforms mating type I cells so that they are able
to form pairs. As a consequence of the interaction,
the cells temporarily and partially fuse and undergo meiosis in the micronucleus, form gametic nuclei, exchange one of the gametic nuclei, form
fertilization nuclei, break down old macronuclei,
form macronuclear anlagen, etc., to complete theconjugation. Conjugation is a significant process
as such in ciliates, however, the details of molecular mechanisms involved in the initiation of conjugation have not yet been elucidated. To reveal theenvironmental factors brought by starvation which
trigger gamone 1 expression, the experimental
system which controls gamone 1 expression isindispensable.
The genus Blepharisma is a suitable material to investigate the mechanisms underlying theinitiation of conjugation for the following two reasons. (1) The mating pheromones gamone 1 andgamone 2 have been isolated and identified(Kubota et al., 1973; Miyake and Beyer, 1974).
The gene encoding gamone 1 was isolated(Sugiura and Harumoto, 2001), and the expressionof the gene can be evaluated at the transcriptional,
protein and biological activity levels (Sugiura etal., 2005). (2) Gamone 1 induces pairs (homotypicpairs) of type II cells at a very low concentration(16 U/ng gamone 1/ml) (1 unit [U] is the smallest
amount of gamone activity that can induce at least
one pair in 500–1000 cells that are suspended in 1
ml of a non-nutrient salt medium) (Miyake, 1981b;
Miyake and Beyer, 1974). The biological activityof gamone 1 secreted even by a single cell can beevaluated.
We previously reported that the transcriptional level of gamone 1 is correlated with the starvation of cells (Sugiura et al., 2005). Gamone 1
transcription does not occur in cells at the logarithmic (log) phase; it occurs at the stationary phase. It
is still unknown whether proliferating cells starttranscribing gamone 1 in response to the sudden
deprivation of a nutrient. If so, how rapid would
the response be? Is gamone 1 expression correlatedwith cell-cycle stage of Blepharisma? To addressthese questions, we here investigated the gamone 1
expression in artificially starved cells and determined the time required for the initiation of transcription and secretion of gamone 1 in Blepharisma japonicum. Our present findings provide anexperimental system to clarify in future the environmental factors for the initiation of conjugation.
MATERIALS AND METHODS
Cells and cell culture
B. japonicum strains R1072 (mating type I)
and R48 (mating type II) were used. Cells were
cultured at 25°C in WGP (Wheat Grass Powder,
Pines) medium (Tokusumi and Takagi, 2000) inoculated with Enterobactor aerogenes 2 days before
use.
Preparation of log-phase and stationary-phasecells and the induction of nutrient depletion
For the preparation of log-phase cells, cells
were filtered through gauze and concentrated bymild centrifugation after 24-h cultivation, and they
were washed three times with physiological balanced solution SMB(−) [Synthetic Medium forJpn. J. Protozool. Vol. 49, No. 1, 2. (2016)

Blepharisma without EDTA (Miyake, 1981b)](designated SMB here) and then suspended in
SMB. For the preparation of stationary-phase cells,
cells were collected after 3–4 days’ cultivation,and similarly suspended in SMB after washing.
The cell density of both log-phase and stationaryphase cell suspension was adjusted at 7000 cells/ml.
Preparation of cell-free fluid (CFF) and the gamone 1 activity assay
A cell suspension of log-phase or stationaryphase type I cells was prepared and kept for theindicated time in SMB (nutrient-depleted condition) at 25°C. After incubation, CFF was obtainedby removing cells from the cell suspension by mild
centrifugation and filtration through a nylon net (5µm × 5 µm). The obtained CFF contained gamone
1, which was secreted by the type I cells during
each incubation time.
The gamone 1 activity is represented in units(U) measured as described (Kubota et al., 1977;
Miyake, 1981b). R48 strain (mating type II) cells
were used as tester cells for the gamone 1 activity
assay.
Isolation of total RNA
Cells were collected by mild centrifugation
after they were incubated in SMB for the indicatedtime. The collected cells were transferred to icecold SMB and incubated for 10 min to remove
pigments (blepharismine), then suspended in TRIzol reagent (Thermo Fisher Scientific, Wilmington, DE). Total RNA was isolated by the acid
guanidinium-phenol-chloroform method as described by Sugiura et al. (2005).
Northern hybridization analysis
To detect gamone 1 mRNA, full-length gamone 1 cDNA was labeled with digoxigenin (DIG)
and used as probes. Total RNA (10 µg/lane) was
fractionated on a 1% agarose gel containing 2.2 M
formaldehyde and transferred to positively charged29

nylon membranes (Roche Molecular Biochemicals, Indianapolis, IN). Membranes were incubatedwith DIG-labeled gamone 1 probe in standard hybridization buffer [5×SSC; 0.1% (w/v) Nlauroylsarcosine; 0.02% (w/v) SDS; 1% blocking
reagent]. After the hybridized membranes were
washed, DIG was detected using a DIG Luminescent Detection kit (Roche Molecular Biochemicals). Details of the method were described by
Sugiura et al. (2005). Transcriptional activity wasevaluated as follows: the intensity of each band
was quantified by the ImageJ (http://
rsb.info.nih.gov/ij/) and normalized with loading
controls (rRNA).
Gamone 1 expression assay in single cells
To examine the starting point of gamone 1
expression in individual cells, we prepared cells
from log-phase cultures and starved them as described below. Log-phase cells were randomly
selected and washed with SMB. A single cell was
isolated to a drop of SMB (1 cell/20 µl SMB/drop)
in a plastic dish. Approximately 24 drops (24 isolated cells) were prepared in a plastic dish. A 50-µl
tester cell suspension (1000–2000 cells/ml)(moderately-starved mating-reactive type II cells)
was then mixed with each drop, and the number ofdrops in which pair formations were observed wascounted at the indicated time points.
To obtain cells shortly after cell division,
dividing cells were isolated from log-phase cultures in WGP. After cell division, the cells were
washed with SMB and a single cell was further
isolated in a drop of SMB on a plastic dish. Theneach cell was tested for the gamone 1 activity as
described above.
For the observation of the frequency of cell
division, randomly selected log-phase cells or cells
just after cell division were isolated to a drop of
SMB (1 cell/70 µl SMB/drop) in a plastic dish.
Approximately 24 drops (24 isolated cells) wereprepared in each sample. The number of drops inwhich the isolated cell divided was counted at the30

Rapid response of gamone 1 expression by starvation

indicated time points.
RESULTS
Gamone 1 expression was induced by sudden
artificial starvation
Mating-type I cells (stock R1072) which
inoculated in the fresh culture medium started proliferating (the culture 1 day after inoculation ishere called log-phase culture), and 3–4 days after
inoculation they reached the stationary phase.
These log-phase and stationary-phase cells weretransferred to the non-nutrient medium (SMB:
Synthetic Medium for Blepharisma) and incubated. CFF was obtained at 30 min (0 day), 1, 3, or 10
days after incubation.
The gamone 1 activity of CFF was evaluatedby mixing the CFF and type II cells as described inMaterials and Methods. The incubated stationaryphase cells (Fig. 1, black bars) started secretinggamone 1 on day 0 (25 U/ml). The gamone 1 activity of incubated cells from the stationary phaseincreased gradually with the incubation time. The
incubated cells from the proliferating cell population (Fig. 1, gray bars) which underwent forced
starvation did not secrete gamone 1 on day 0, butthey started secreting it by day 1 (211 U/ml). The
gamone 1 activity increased with the incubation
time. Incubated cells from both the stationary
phase and the log phase showed approximately thesame gamone 1 activity at 1, 3 and 10 days.
The time required for the expression of gamone 1
induced by sudden artificial starvation
Proliferating log-phase cells incubated in
SMB secreted gamone 1 within 1 day. We theninvestigated the time required for the initiation oftranscription and the secretion of gamone 1 in incubated cells from the log phase in more detail.
We tested CFF obtained from incubated cells in
SMB at 2, 3, 4, 5, 8, 11, 14 and 26 h for gamone 1
activity (Fig. 2A, graph). At the start of the test (2h), gamone 1 activity was not detected in CFF

Fig. 1. Gamone 1 expression induced by artificial starvation. After the inoculation of cells to the fresh culture
medium, cells were transferred to a non-nutrient medium(SMB) on the 1st day (log-phase cells) or 3rd day(stationary-phase cells) and incubated for the indicatedtime. The CFF obtained was bioassayed for gamone 1
activity. Gamone 1 activity was measured twice in eachtime point. Ordinate: the gamone 1 activity (n) (2 n U/ml).
Mean ± s.d. Abscissa: the number of days after transfer ofcells to SMB. Black bar: cells obtained from stationary
phase. Grey bar: cells obtained from the log phase.

indicating that the incubated cells from the proliferating cell population did not secrete gamone 1
within 2 h. These cells started secreting gamone 1
within 3 h and the activity gradually increased to
213 U/ml at 26 h (Fig. 2A, graph).
The transcriptional activity of gamone 1 gene
was investigated in incubated cells from log phase(Fig. 2A, photo). Total RNA was isolated, northernhybridization was probed with gamone 1 cDNA,and the transcriptional activity was evaluated as
described in Materials and Methods. After thetransfer of log-phase cells to the non-nutrient medium (SMB), gamone 1 transcription was slightly
detected at 2 h (arrow, Fig. 2A, photo)(transcriptional activity: 0.12), and the activity had
greatly increased (51.8-fold) by 26 h(transcriptional activity: 6.22).
As gamone 1 transcription started within 2 h
at most after the transfer of log-phase cells to the
non-nutrient medium, we investigated the gamone
1 activity in the external medium and gamone 1
transcription in incubated cells from the log phaseJpn. J. Protozool. Vol. 49, No. 1, 2. (2016)31

1 activity was not detected in the external medium
at 0.5–2.5 h but detected by 3.5 h (Fig. 2B, graph).
These results indicate that the cells started the transcription of gamone 1 gene at approx. 1.5 h andsecrete gamone 1 at 3.0–3.5 h after the transfer tothe non-nutrient medium.
Both gamone 1 transcriptional activity andgamone 1 activity in the external medium increased with time after the sudden deprivation ofnutrients in incubated cells from the log phase.
Two possible alternative scenarios explaining why
the activities increased with time are as follows.
(1) All of the cells in the population consistently
and uniformly show the expression of gamone 1.
The gamone 1 expression per cell is low at the
beginning and gradually increases, and then thegamone 1 activity of the population increases withtime, or (2) the cells in the population are not all
consistent regarding the expression of gamone 1,
as the population is a mixture of cells with andwithout gamone 1 activity. At the beginning, only
a few cells have the activity, and the ratio of thecells with activity increases with time. To elucidate which of these scenarios is correct, we evaluated the gamone 1 activity of individual cells.
Fig. 2. The time required for the expression of gamone 1
induced by sudden artificial starvation in cells taken fromthe log-phase. Log-phase cells were transferred into SMB
and incubated for 2, 3, 4, 5, 8, 11, 14 and 26 h (A), or 0.5,
1, 1.5, 2, 2.5, 3.5, 4.5 and 6.5 h (B). At each incubation
time, the biological activity of gamone 1 released in CFF
was estimated (Graphs), and transcriptional activity of thegamone 1 gene was assayed by Northern hybridization
analysis (Blots). (Graphs) Ordinate: the gamone 1 activity(n) (2n U/ml). Mean ± s.d. Abscissa: the time (hours: h)after the transfer of cells to SMB. (Blots) Northern blots
were probed with cDNA of gamone 1. G1: gamone 1 transcript (1000 bp). rRNA: ethidium bromide-stained loading
control. Arrow marks the detection of the transcript at 2 h(A) and 1.5 h (B).

every 30 min starting 0.5–2.5 h after the transfer(Fig. 2B). The arrow in the photo shows that gamone 1 transcription was not initiated at 0.5 and 1 h
but was initiated by 1.5 h after the transfer of logphase cells into the non-nutrient medium. Gamone

Gamone 1 expression in individual cells inducedby sudden artificial starvation
We isolated proliferating type I cells andmixed them with tester cells (moderately starved
type II cells: stock R48) in a small drop of SMB as
described in Materials and Methods (Fig. 3A). The
drops were observed if mating pairs were formed
according to time (h) after mixing with tester cells.
If an isolated type I cell secretes gamone 1, tester
cells would form homotypic pairs. The ratio ofcells that secrete gamone 1 was considered here as
the ratio of the drops in which pair formation wasobserved among 22 or 24 drops.
In randomly isolated log-phase cells, gamone
1 secretion was first detected at 3 h, and the ratioof cells that secreted gamone 1 was zero at 0 h,
0.18 at 5 h, 0.27 at 10 h and 0.95 at 24 h (Fig. 3B).

32

Rapid response of gamone 1 expression by starvation

Fig. 3. Gamone 1 expression in single cells induced by sudden artificial starvation. (A) Scheme for the experimental
design. Proliferating type I cells were isolated, transferred into a small drop of SMB, and mixed with tester cells(moderately-starved mating-reactive type II cells). The 22–24 drops (70 µl each) were incubated and observed for mating pairs. (B, C) Ordinate: the ratio of the drops in which pair formation was observed in 22 or 24 drops. Abscissa: thetime after the cells were mixed. (B) Type I cells were randomly isolated from a proliferating cell population, and (C)type I cells shortly after division were isolated. Mean ± 95% confidence limits.

The number of cells that secreted gamone 1 increased with time, but it should be pointed out thateven at 10 h, <30% cells secreted gamone 1. This
result suggests that the individual cells were notsynchronized for gamone 1 expression but rather
had different timings for the expression.
The proliferating cell culture likely consistsof cells at various cell-cycle stages. It is plausiblethat the time required for gamone 1 expression
depends on the cell-cycle stages in which the isolated cells were placed. These results are consistent with the idea that the isolated cells startgamone 1 transcription and secretion when thecells undergo the cell cycle and enter a certain
stage in which gamone 1 transcription is possible.
To investigate the correlation between gamone 1 expression and the cell cycle, we isolated

cells shortly after division from proliferating logphase cultures and tested them for gamone 1 expression (Fig. 3C). We compared these results withthose of the randomly isolated cells from proliferating log-phase cultures (Fig. 3B). The gamone 1
expression of the isolated cells shortly after division showed a profile that differed from that of therandomly isolated cells. Fig. 3C shows that theratio of cells that secreted gamone 1 increased withtime from zero at 0 h to 0.58 at 5 h, 0.88 at 10 h,
and 0.92 at 24 h. Thus, the gamone 1 expression
began sooner in the isolated cells shortly after division than in randomly isolated cells. This result
suggests that the individual cells shortly after division were relatively synchronized in the cell cycle,
and started expressing gamone 1 at about the sametime. Our next experiment was conducted to test

Jpn. J. Protozool. Vol. 49, No. 1, 2. (2016)33of the cells completed division by 24 h (Fig. 4A,line graph). In contrast, in the isolated cells shortly
after division, the ratio of cells expressing gamone
1 increased from 5 h and reached 0.6 at 12 h and
1.0 at 24 h (Fig. 4B, bar graph). Cell division was
not observed by 24 h, and <10% cells had divided
at 36 h (Fig. 4B, line graph). These data demonstrated that randomly isolated cells gradually divided from 10 h, and only a few cells expressedgamone 1 by 12 h, whereas the cells shortly after
division did not divide by 24 h and started expressing gamone 1 earlier.
DISCUSSION

Fig. 4. Correlation between cell division and gamone 1
expression induced by sudden artificial starvation in single
cells. Proliferating type I cells were isolated, transferred
into a small drop of SMB, mixed and incubated with tester
cells (moderately-starved mating-reactive type II cells) or
incubated without mixing with tester cells. The 24 drops(70 µl each) mixed with tester cells were observed formating pairs and the drops incubated without mixing withtester cells were observed for cell division according to theindicated time. (A) Type I cells were randomly isolated
from a proliferating cell population, and (B) type I cells
shortly after division were isolated. Ordinate: the ratio ofdrops in which mating pairs were observed (bars), and theratio of cells that divided (lines). Mean ± 95% confidence
limits. Abscissa: the incubation time (hours).

the correlation between gamone 1 expression andcell division.
We investigated the time required for gamone 1 expression and cell division in individual
isolated cells taken randomly and in those takenfrom a divided cell population. Among the randomly isolated cells at 10–12 h, <10% expressedgamone 1, and nearly 80% of the cells expressedgamone 1 at 24 h (Fig. 4A, bar graph). Cell division was first observed at 10 h, and approx. 30%

Fig. 1 indicates that the log-phase cells
which did not secrete gamone 1 could rapidly respond to the artificial sudden starvation, start secreting gamone 1, and reach the same level ofgamone 1 expression as in cells from the stationary-phase within 1 day. Fig. 2 indicates that gamone 1
transcription started about 1.5 h at most after thetransfer of log-phase cells to the non-nutrient medium, and the transcriptional activity increased
with time. Gamone 1 secretion was observed at 3 h
in the external medium, and it also increased withtime. Figs. 1 and 2 revealed that gamone 1 expression (transcription and secretion) is induced not
only by gradual starvation from the log phase to
the stationary phase, but also by a sudden deprivation of nutrients from log-phase cultures. An approx. 1.5-h incubation in the non-nutrient medium
was required for the initiation of transcription ofgamone 1 gene. Our findings clearly show that thegamone 1 transcription and secretion are strictly
controlled by the environmental nutrient condition,
as predicted in our previous report (Sugiura et al.,2005). These results also indicate that proliferating
cells can be transformed to gamone-producing
cells by artificial starvation, which enables to provide a useful experimental system to investigate
the environmental factors to trigger conjugation in
B. japonicam.

34

Rapid response of gamone 1 expression by starvation

Fig. 3 showed that in the randomly isolated
log-phase cells, the time required for gamone 1
expression was various; from 3 h to about 24 h,while in the cells shortly after division, the time
was relatively synchronized; from 3 h to10 h. The
time required for gamone 1 expression was relatively shorter in cells shortly after division was
also shown in Fig. 4. The Figure indicates that in
the randomly isolated log-phase cells, the timerequired for cell division varied; the shortest was
10 h, and in the isolated cells shortly after division,
the time was longer than 24 h. Together these findings, we suggest that in randomly isolated logphase cells, some cells express gamone 1 only
after cell division, and cell-cycle progression isnecessary in these cells for gamone 1 expression.
In cells shortly after division, we speculate that allcells can express gamone 1 without cell division.
Our present results strongly indicate that gamone 1
expression is correlated with the cell cycle of B.
japonicum. The cell cycle of Blepharisma intermedium (later classified as Blepharisma japonicum
var. intermedium) was reported as approximately
16 h of G1, 7 h of S, and 1–2 h of G2 phase(Minutoli and Hirshfield, 1968). We suggest thatcells start expressing gamone 1 at the certain pointof the cell cycle, within several hours after cell
division possibly at the G1 phase.
Fig. 5 shows a putative scheme of the correlation between gamone 1 expression and the cell
cycle. Our present findings suggest that gamone 1
expression starts at a certain stage of the cell cycle(most probably G1 phase), and artificial starvationinduces gamone 1 expression depending on thecell-cycle stage in which the cell is placed. For
example, the cell at stage (a) in Fig. 5 (shortly after
division) becomes able to express gamone 1 withinseveral hours without subsequent division. A cellat (b) which is in putative G1 needs about 1.5 h for
initiation of the transcription of gamone 1. The
cells at (c) and (d) which are placed at more advanced stage do not express gamone 1 and takesome time to reach the subsequent division stage,

Fig. 5. The cell cycle and gamone 1 expression: A presumptive scheme. A presumptive cell cycle of Blepharisma is shown according to Minutoli and Hirshfield (1968).
The present findings suggest that gamone 1 expression
starts at a certain stage of the cell cycle (most probably G1phase), and sudden artificial starvation induce gamone 1
expression depending on the cell-cycle stage in which thecell is placed. The cell at (a) shortly after division becomes able to express gamone 1 within several hours
without subsequent division. A cell at (b) needs about 1.5
h for initiation of the transcription of gamone 1. The cells
at (c) and (d) do not express gamone 1 and take some time
to reach the subsequent division stage, D, and they express
gamone 1 after cell division.

and express gamone 1 only after cell division. If
cells are isolated randomly from a proliferatingcell population (Figs. 3B and 4A), the cells are invarious stages of the cell cycle, and the time required for gamone 1 expression would depend on
every cell stage. If cells shortly after division are
isolated (Figs. 3C and 4B), all of the cells would
be at (a) (Fig. 5), and they express gamone 1 without subsequent division which is consistent withthe results.
In ciliates, conjugation usually occurs whencells are moderately starved. In Paramecium
tetraurelia, the putative mating type-determining
gene, mtA, was recently identified (Singh et al.,2014), and northern blot analyses confirmed that it
is expressed mating-type specifically and only inJpn. J. Protozool. Vol. 49, No. 1, 2. (2016)

moderately starved, sexually reactive E cells. The
expression is limited to sexually reactive cells because transcripts could not be detected in exponentially growing cells or in over-starved cells. In
Tetrahymena thermophila, expression of the mating type-specific gene was observed only in moderately starved mating-compatible cells, not in
exponentially growing cells (Cervantes et al.,2013).
In Blepharisma japonicum, the present investigation clearly revealed that the transcription andsecretion of the mating pheromone, gamone 1 isstrictly correlated with starvation. Proliferating
Blepharisma cells rapidly responded to sudden
artificial starvation by the initiation of transcription and the secretion of gamone 1, in a relatively
short time, approx. 1.5 h and 3 h, respectively. The
results of our study also provide an evidence thatthe gamone 1 expression is correlated with a certain stage of cell-cycle. In Euplotes raikovi, mating pheromones are expressed throughout the cell
cycle regardless of the log or stationary phase, andthe mating pheromone is also involved in the proliferation of cells (Luporini et al., 2006; Vallesi etal., 1995). It is still unknown why these differences
occur, and the regulation of gene expression insuch mating type-specific genes, mating pheromones, or gamones may differ among species.
The deprivation of a nitrogen source combined with the presence of a nonfermentable carbon source leads cells to enter the developmental
pathway of meiosis and sporulation in budding
yeast Saccharomyces cerevisiae (Freese et al.,1982). The trigger molecules involving the initiation of conjugation in Blepharisma are unknown
yet, however, our study provides an experimental
system to reveal the environmental factors by theexperiment e.g. adding certain nutrient into theartificially starved cells to inhibit gamone 1 transcription. The identification of the factors is now
under investigation.

35

ACKNOWLEDGEMENTS
We appreciate Prof. Emeritus of Nara Women’s University, Yoshiomi Takagi, for useful discussions. This study was supported by Grants-inAid for Scientific Research from the Japan Societyof the Promotion of Science to M.S. (No.
26440126) and T.H. (No. 25440210), and by Nara
Women’s University Intramural Grant for Project
Research (2015) to M.S.
REFERENCES
Cervantes, M. D., Hamilton, E. P., Xiong, J., Lawson, M. J., Yuan, D., Hadjithomas, M., Miao,
W. and Orias, E. (2013) Selecting one ofseveral mating types through gene segmentjoining and deletion in Tetrahymena thermophila. PLOS Biol., 11, e1001518.
Freese, E. B., Chu, M. I. and Freese, E. (1982)
Initiation of yeast sporulation of partial carbon, nitrogen, or phosphate deprivation. J.
Bacteriol., 149, 840–851.
Kubota, T., Tokoroyama, T., Tsukuda, Y., Koyama, H. and Miyake, A. (1973) Isolation andstructure determination of blepharismin, aconjugation initiating gamone in the ciliate
Blepharisma. Science, 179, 400–402.
Kubota, T., Miyake, A. and Tokoroyama, T.
(1977) Isolation and purification of conjugation-inducing substances in Blepharisma. In:
Experimental Methods on Natural Organic
Compounds - Extraction and Isolation of
Physiologically Active Substances. Natori,
S., Ikekawa, N. and Suzuki, M. (eds.). Kodansha, Tokyo, pp. 485–498.
Luporini, P., Vallesi, A., Alimenti, C. and Ortenzi,
C. (2006) The cell type-specific signal proteins (pheromones) of protozoan ciliates.
Curr. Pharm. Des., 12, 3015–3024.
Miyake, A. (1981a) Physiology and biochemistryof conjugation in ciliates. In: Biochemistry
and Physiology of Protozoa. Levandowsky,

36

Rapid response of gamone 1 expression by starvation

M. and Hutner, S. H. (eds.). Academic Press,
New York, pp. 125–198.
Miyake, A. (1981b) Cell interaction by Gamonesin Blepharisma. In: Sexual Interactions inEukaryotic Microbes. O’Day, D. H. andHorgen, P. A. (eds.). Academic Press, New
York, pp. 95–129.
Miyake, A. (1996) Fertilization and sexuality inciliates. In: Ciliates: Cells as Organisms.
Hausmann, K. and Bradbury, P. C. (eds.).
Gustav Fischer Verlag, New York, pp. 243–
290.
Miyake, A. and Beyer, J. (1974) Blepharmone: A
conjugation inducing glycoprotein in theciliate Blepharisma. Science, 185, 621–623.
Minutoli, F. and Hirshfield, H. I. (1968) DNA
synthesis cycle in Blepharisma. J. Protozool.,
15, 532–535.
Singh, D. P., Saudemont, B., Guglielmi, G., Arnaiz, O., Goût, J. F., Prajer, M., Potekhin, A.,
Przybòs, E., Aubusson-Fleury, A., Bhullar,
S. et al. (2014) Genome-defence small RNAs
exapted for epigenetic mating-type inher-

itance. Nature, 509, 447–452.
Sugiura, M. and Harumoto, T. (2001) Identification, characterization, and complete aminoacid sequence of the conjugation-inducing
glycoprotein (blepharmone) in the ciliate
Blepharisma japonicum. Proc. Natl. Acad.
Sci. U.S.A., 98, 14446–14451.
Sugiura, M., Kawahara, S., Iio, H. and Harumoto,T. (2005) Developmentally and environmentally regulated expression of gamone 1: thetrigger molecule for sexual reproduction in
Blepharisma japonicum. J. Cell Sci., 118,
2735–2741.
Tokusumi, Y. and Takagi, Y. (2000) Ectosymbiotic role of food bacteria for Paramecium:
bacterial detoxification of paramecia-killing
toxin contained in wheat grass powder. Zool.
Sci., 17, 341–348.
Vallesi, A., Giuli, G., Bradshaw, R. A. and Luporini, P. (1995) Autocrine mitogenic activity of pheromone produced by the protozoan
ciliate Euplotes raikovi. Nature, 376, 522–
524.