Jpn. J. Protozool. Vol. 40, No. 2. (2007)

131

Original
Cdk1 and cks gene homologs are transcriptionally activated during
induction of conjugating pairs in mating-type II cells of the ciliate
Blepharisma japonicum

Yuri TANAKA1, Mayumi SUGIURA2 and Terue HARUMOTO3*
1

Department of Biological Science and Environment, Graduate School of Human Culture, Nara
Women's University, Kitauoya-nishi-machi, Nara, 630-8506, Japan. 2Department of Biology, Graduate
School of Science, Kobe University, Kobe 657-8501, Japan, JSPS Research Fellow (PD). 3Departmentof Biological Science, Faculty of Science, Nara Women's University, Kitauoya-nishi-machi, Nara, 6308506, Japan.
Key words: gene expression, conjugation, subtraction, mating pheromone

SUMMARYIn Blepharisma japonicum, conjugation is
induced by interaction between cells of complementary mating-types I and II. Cells of bothmating-types produce and secrete mating pheromones (gamones). Cells that have received complementary gamones undergo morphologicalchanges (rounding) and start to unite. Nuclear
changes, including meiosis, gametic nuclei*Corresponding author
Tel: + 81 742 20 3421
Fax: + 81 742 20 3421
E-mail: harumoto@cc.nara-wu.ac.jp
Received: 22 April 2007; Accepted: 11 May 2007.
Note: Nucleotide sequence data reported are available
in the DDBJ/EMBL/GenBank databases under theaccession numbers from AB325669 to AB325675.

exchange, fertilization and development of new
macro- and micronuclei occur in the conjugants.
Although conjugation is such a striking
phenomenon, the molecular mechanism of
induction of conjugation remains unknown. In
order to identify genes that are involved in formation of conjugating pairs, we isolated genes thatwere expressed specifically in conjugationinduced type II cells, using suppression subtractive hybridization. To induce conjugation, we
treated type II cells for 4 hours with a cell-free
fluid from type I cells which contained gamone1.
During this period, type II cells formed pairs, although these homotypic pairs never entered meiosis. We then purified polyA+RNA and subjected it
to cDNA synthesis. This cDNA was then subtracted with cDNA that was prepared from untreated cells. We obtained eight gene fragments.
Homology searches revealed that three of these

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Genes activated during conjugation in Blepharisma

fragments showed significant homology to the cdk
family (cdk1 and cdk2), 4-hydroxy-phenylpyruvate
dioxygenase (4-HPPD) and cyclin dependent
kinase regulatory subunit (cks). Northern hybridization demonstrated that these three genes were
specifically transcribed in cells treated with gamone1. We also found that the transcripts had already appeared 2 hours after the onset of gamone1
treatment. Cdk1 and cks are generally involved incell-cycle regulation, but are here specifically expressed during induction of homotypic type II
pairs that undergo neither mitosis nor meiosis.

INTRODUCTION
Conjugation of the heterotrich ciliate Blepharisma japonicum is induced by the interaction oftype I and type II complementary mating-type cells(Miyake, 1968; Miyake and Beyer, 1973). Vegetative cells continuously divide under nutrient-richconditions. However, when deprived of nutrients,
cells begin sexual reproduction (conjugation).
First, type I cells start secreting gamone1, one of
the conjugation-inducting substances (mating
pheromones), and this stimulates type II cells tosecrete gamone2, the other conjugation-inducing
substance. Gamone2 promotes gamone1 secretionin type I cells. Both mating-type cells are transformed, ready to join and enter the sexual reproduction process (Miyake, 1981, for review).
Gamone1 and gamone2 have both been isolated
and characterized. Gamone1 is a glycoprotein of
about 30 kD, and consists of 272 amino acids andseven sugar residues (Miyake, 1981; Sugiura andHarumoto, 2001; Harumoto and Sugiura, 2003).
Gamone2 has been identified as calcium-3-(2'formylamino-5'-hydroxybenzoyl) lactate, which
can be chemically synthesized (Kubota, et al.,1973).
Treatment with cycloheximide inhibits conjugating pair formation, which indicates that new

protein synthesis is essential for the pair formation(Miyake and Honda, 1976). This suggests expression of various genes during this peroid, but it
has not been known which genes are activated bygamones and which genes are involved in the formation of conjugating pairs.
In this study, we used suppression subtractive
hybridization in type II cells to isolate genes expressed during induction of conjugation. Suppression subtractive hybridization is an effective
method for isolating genes that are differentiallyexpressed in two conditions (Diatchenko, et al.,
1999; Fetzer, et al., 2002). Gene expression wascompared between conjugation-induced and noninduced cells, and the genes which expressed specifically in gamone1-treated cells were identified.
Type II cells are suitable for isolating such genes,
because whereas type I cells begin secreting gamone1 autonomously in response to starvation,
type II cells usually do not respond this way. They
start synthesizing gamone2 and progress toward
conjugation only after being stimulated by gamone1. The cDNAs of type II cells before andafter treatment with gamone1 were subjected tosubtraction, and eight gene fragments were isolated. Homology searches revealed that three ofthese fragments are homologous to cyclin dependent kinase 1 (cdk1), cyclin dependent kinase regulatory subunit (cks) and 4-hydroxyphenylpyruvate
dioxygenase (4-HPPD). We confirmed that thesegenes are expressed only in type II cells stimulated
by gamone1 by northern hybridization. This unusual expression of cdk1 and cks homologs in conjugating pairs of Blepharisma may indicate a novel
function for these genes.

MATERIALS AND METHODS
1. Blepharisma stocks and culture method
We used mating-type II cells of Blepharisma
japonicum R48 strain. The cells were cultured inJpn. J. Protozool. Vol. 40, No. 2. (2007)

diluted extract of wheat grass powder (Pines) withphosphate buffer (pH 6.8) and stigmasterol (5×10-4
mg/ml) at 25°C (Tokusumi and Takagi, 2000).
Enterobacter aerogenes was inoculated, andcultured for 2 days before use. The method of culturing was described in detail by Harumoto andSugiura (2003).
2. Gamone1 treatment for type II cells
Mating type II cells (1.2×106) were collectedby centrifugation at 100×g and suspended for one
day in physiologically balanced solution (modified
synthetic medium for Blepharisma, SMB; Miyake,
1981; Harumoto and Sugiura, 2003) consisting of
1.5 mM NaCl, 0.05 mM KCl, 0.4 mM CaCl2,
0.05 mM MgCl2, 0.05 mM MgSO4 and 2.0 mM
phosphate buffer (pH 6.8). The cell suspension(1,500‒2,000 cells/ml) was divided into two samples; one was treated with cell-free fluid fromtype I cells (CFF1; 103 U/ml gamone1) (Miyake
and Beyer, 1973; Miyake, 1981), and the other was
not treated. Cells were treated with CFF1 for
4 hours to prepare samples for subtraction andnorthern hybridization, and for 2 or 4 hours for dot
hybridization.
Frozen CFF1 was thawed and used in this study.
Preparation of CFF1 was described in Sugiura andHarumoto (2001).
3. Total RNA and polyA+RNA isolation
Total RNA was isolated from both gamone1treated and untreated cells. Blepharisma has pigment granules under the cell surface (Giese, 1973),
which often prevent recovery of nucleic acids from
cells. To remove this pigment, Blepharisma cells
were subjected to cold shock treatment with SMB
at 4°C for 10 minutes. After this treatment, cells
discharged massive pigment granules.
Five milliliters of TRIzol Reagent (Invitrogen)was added to about 1×105 cells, and total RNA
was isolated by the acid guanidinium–phenol–
chloroform method. Total RNA isolated from a

133

3,000-ml culture was 1 mg. PolyA+RNA was isolated with the Oligotex-dT30 [SUPER] kit (Roche
Molecular Biochemicals). PolyA+RNA was concentrated by ethanol precipitation and adjusted to aconcentration of 1 µg/µl.
4. Suppression subtractive hybridization
Suppression subtractive hybridization was used toisolate differentially expressed transcripts. Firstand second strand cDNA synthesis, RsaI endonuclease enzyme digestion, adapter ligation, hybridization and PCR amplification were performed
according to the manufacturer's protocol for the
PCR Select cDNA subtraction kit (Clontech). Subtracted and unsubtracted PCR products were run
on a 2% agarose gel to observe the differential
band pattern. Specific bands in the subtracted sample were excised, gel-extracted and cloned into
pCR2.1-TOPO vector (Invitrogen).
5. Sequencing and homology search
Sequencing was performed by either of the following methods: 1) Big dye terminator v3.1 cycle
sequencing kit (Applied Biosystems); the samples
were sequenced using ABI3100. 2) Thermo sequence cycle sequencing kit (Amersham Biosciences); sequencing done by DSQ-2000L(Shimadzu). The determined sequences were subjected to homology search through the BLAST
server provided by DNA Data Bank of Japan(DDBJ, http://blast.ddbj.nig.ac.jp/top-j.html).
6. Northern hybridization and dot hybridization
Probes were synthesized with a DIG DNA labeling kit (Roche Molecular Biochemicals). In northern hybridization, 10 µg total RNA was subjected
to electrophoresis on denaturing 1% agarose gel
and blotted onto positively charged nylon membranes (Roche Molecular Biochemicals). In dot
hybridization, 5–10 µg total RNA was dropped
directly on the membranes.

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Genes activated during conjugation in Blepharisma

Table 1. Homology search for eight cDNA fragments obtained by suppression subtractive hybridizationin this study.
fragment
size

BLAST search result
protein homolog

E-values1001

432 bpCdc2 cyclin dependent kinase (Pneumocystis carinii, 104-246)*

3 × e-50

s1002

140 bpno match domains1003

878 bpElongation factor 1 gamma-3, EF-1-γ3(Oryza sativa, 149-304)*

1 × e-8s1004

710 bpHypothetical protein (Plasmodium berghei, 73-201)*

1.8

s1005

526 bpHypothetical protein (Emericella nidulans, 158-244)*

0.82

s1006

343 bpGlyceraldehyde-3-phosphate dehydrogenase protein(Blepharisma japonicum, 228-303)*

3 × e-35

s1007

258 bpPutative 4-hydroxyphenylpyruvate dioxygenase, 4-HPPD(Paramecium tetraurelia, 179-262)*

4 × e-31

s1008

227 bpCyclin dependent kinase regulatory subunit, cks(Tetrahymena thermophila, 45-96)*

4 × e-11*Species with highest score is denoted. Numbers indicate amino acid residues that matched this sequence.

RNAs were UV-crosslinked to the membrane,
and then hybridized with DIG-labeled DNA
probes. The probes were detected by DIG luminescent detection kit (Roche Molecular Biochemicals).

RESULTS
Subtractive hybridization, sequencing and homology search of genes isolated from cells
treated with gamone1
To isolate genes expressed in type II cells of B.
japonicum during the induction of conjugating pairformation by gamone1, we used the suppression
subtractive hybridization method. The type II cells
had started to form homotypic pairs 2 hours after
the addition of CFF1. Fewer than 10% of cells had
paired at 2 hours, but the number gradually increased. After 4 hours, most cells had been activated and formed pairs. Suppression subtractive
hybridization was performed using cDNAs createdfrom polyA+RNA isolated from gamone1-treated(4 hr) cells as the ‘tester’ and from untreated cells
as the ‘driver’ (Clontech). Subtracted cDNA fragments were amplified by PCR and electrophoresed. Eight major bands with lengths ranging between about 200 and 900 bp were gel-extracted,
cloned and sequenced. One of these bands contained two individual sequences (s1007, s1008).
Among these nine sequences, a part of one sequence matched another sequence (s1001). We
eventually obtained eight distinct sequences fromsuppression subtractive hybridization (s1001 ‒s1008).
Homology searches revealed that one of thesequences(s1006)was
glyceraldehyde-3phosphate dehydrogenase (GAPDH), a gene already known in B. japonicum. The other seven
sequences (s1001–s1005, s1007, s1008) were
newly isolated in B. japonicum. Table 1 shows theresult of homology searches for each sequence.
Fragment s1001 had a length of 432 bp andwas deduced to encode 143 amino acids (aa). A

Jpn. J. Protozool. Vol. 40, No. 2. (2007)

search revealed high homology to cyclin dependent kinase, cdk1 (cdc2) or cdk2 in a wide range oforganisms. The gene that showed highest similarity (60% identities, E-value: 3e-50) was cyclin
dependent kinase (cdc2) of Pneumocystis carinii.
Fragment s1007, a 258 bp (85 aa) sequence,
showed high homology to 4-hydroxyphenylpyruvate dioxygenase (4-HPPD).
Fragment s1008, a 227 bp (71 aa) sequence,
showed homology to cyclin dependent kinase
regulatory subunit (cks). Alignment of the sequence with human, Xenopus and other cks homologs revealed that this sequence had a 32residue overhang at the C-terminus of these known
cks proteins. The sequence contains the HXPEPH
motif which is conserved in cks-family proteins.
The sequence of s1003 was 878 bp, an estimated 169 amino acid residues. This sequence
showed a low homology to elongation factor 1
gamma 3 (EF-1-γ3) of Oryza sativa.
Sequence s1002 was 140 bp, consisting of repeated GAA. Sequence s1004 was 710 bp, andshowed low homology to a hypothetical protein of
Plasmodium berghei (73–201 aa); and sequences1005 was 526 bp with low homology to a hypothetical protein of Emericella nidulans.
Confirmation of differential expression by dot
hybridization analysis
To confirm whether these eight genes were expressed specifically in the formation of conjugating pairs, dot hybridization was performed using
total RNA from untreated cells and cells that had
been treated with gamone1 for 2 or 4 hours (Fig.
1). Probes were made from the eight sequences(s1001–s1008). Differential expression signals
were detected only in gamone1-treated samples
probed with s1001 (cdk1 homolog), s1007 (4HPPD homolog) and s1008 (cks homolog). It wasalso shown that these three genes were alreadytranscribed 2 hours after the onset of the gamone1
treatment. No difference in expression was de-Probes135―

24 (hr)

s1001
s1002
s1003
s1004
s1005
s1006
s1007
s1008

Fig. 1. Dot hybridization performed using total RNA
from untreated cells (-), and cells treated for 2 hours (2)and 4 hours (4) with cell-free fluid from type I cells(CFF1). Probes are eight individual cDNA fragments(s1001–s1008) obtained by suppression subtractive hybridization (Table 1). Amounts of applied total RNA were
5 µg for s1002, s1003 and s1006, and 10 µg for s1001,
s1004, s1005, s1007 and s1008.

tected between treated and untreated samples
probed with s1002, s1003, s1004, s1005 ands1006.
Northern hybridization analysis
To analyze the transcripts of cdk1 (s1001) andcks (s1008), total RNA from gamone1-treated anduntreated samples were electrophoresed, blotted
onto nylon membranes, and hybridized withprobes (Fig. 2a and 2b). Ethidium bromide staining
showed that the amount of RNA applied to the gel
was equal. Single bands were detected only intreated samples for both s1001 and s1008. The
sizes
of
the
transcripts
were
about
1,000 nucleotides (s1001, Fig. 2a) and about

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Genes activated during conjugation in Blepharisma

Fig. 2 Northern hybridization
probed with s1001 (cdk1 homolog, a) and s1008 (cks homolog, b). Total RNA (10 µg)
from untreated cells (-) andcells treated with CFF1 for
4 hours (4) were subjected toelectrophoresis and stainedwith ethidium bromide (lefthand panels), and hybridized
with probes (right-hand panels). Arrowheads show positions of detected signals. The
size of RNA (nt; nucleotides) isshown on the left.

500 nucleotides (s1008, Fig. 2b). Most cdk1- andcdk2-family proteins are about 280–300 aa. The
length of the s1001 transcript detected in this study
is consistent with this. When the 500-nucleotides1008 transcript is translated as a protein with
about 150 aa, it is atypically long compared withconventional cks-family proteins (about 100 aa).
The 23-residue overhang at the C-terminus ofs1008, described above, may be a possible explanation.
DISCUSSION
Using suppression subtractive hybridization, we
identified three genes that were expressed specifically in the formation of conjugating pairs oftype II cells of B. japonicum. Cdk1, cks and 4HPPD homologs were isolated for the first time in
B. japonicum. Cdk1 is a protein involved in cellcycle regulation. Cks is a small conserved proteinthat interacts with cdk, contributing to a family of
essential components of the cdk complex that
regulates cell-cycle progression.
In this study, suppression subtractive hybridization was applied to gamone1-treated and untreated type II cells. Type II cells are suited to thismethod because they only start secreting gamone2

and progress toward conjugation after they are
stimulated with gamone1. Type II cells were activated and formed conjugating pairs of the samemating type (homotypic pairs). Unlike heterotypicpairs (type I–type II pairs), homotypic pairs(type II–type II) undergo neither meiosis nor nuclear exchange, and macro- and micronuclei remain unchanged. In B. japonicum, doublet cellswith two mouths have been constructed by treatment with actinomycin S3 (Miyake, 1975). The
doublet can join with two cells, one on eitherside. Complementary gamone treatment induced achain consisting of several doublets. Such homotypic chains did not undergo nuclear changes, but
meiosis was initiated when a single complementary mating-type cell joined at the end of the chain(Miyake, 1975). Meiosis first started in the cellthat united with a complementary cell, and thenpropagated through the chain. This observationsuggests that the nuclear changes are arrested before the onset of meiosis in homotypic pairs, andthe signal produced in a heterotypic pair was transmitted along the chain and released the interruption. It also suggests that homotypic pairs areready for conjugation except for a signal provided
by complementary mating-type cells.
Using type II homotypic pairs, we attempted toJpn. J. Protozool. Vol. 40, No. 2. (2007)

isolate genes involved in the formation of conjugating pairs; for example, genes related to gamone2 biosynthesis, those involved in signal transduction, or those that regulate morphologicalchanges leading to conjugation. We found that
cdk1, cks and 4-HPPD homologs were activatedby gamone1 in type II cells. Expression of additional genes may also be involved in these processes. These genes might be lost during the procedure of subtractive hybridization or be expressed at
levels too low to detect. Significant differential
expression is evident in cdk1, cks and 4-HPPD
homologs, which suggests that they are essential
for the processes induced by gamone1 in type II
cells. It is known that 4-HPPD is involved inamino acid metabolism. Gamone2 is a small molecule which is synthesized from tryptophan(Miyake, 1981). The 4-HPPD homolog isolated inthis study might be involved in the biosynthetic
pathway of gamone2.
It is remarkable that, although cdk1 and cks arecell-cycle related genes, homologs isolated in thisstudy were activated in homotypic pairs which
underwent neither mitosis nor meiosis. In Tetrahymena thermophila, a cdk1 homolog was associated with basal body domains, and knockout of thegene resulted in decondensation of macro- andmicronuclear chromosomes. Zhang, et al. (2002)
suggested that the cdk1 homolog might be involved in establishing morphogenetic pattern formation and also be associated with linking of histone H1. In Paramecium tetraurelia, two cdk1
gene homologs were isolated (Tang, et al., 1995;
Zhang and Berger, 1999). In both T. thermophila
and P. tetraurelia, these genes were expressed
during the mitotic cell cycle. The cdk1 homolog
isolated in this study has relatively low homology
to these cdk1 genes, and there has been no reportthat cdk1 is related to the formation of conjugating
pairs in ciliates. The cdk1 and cks homologs isolated in this study may serve unconventional functions.

137

Cks has been reported to interact geneticallyand physically with cdk. The function of cks as acell-cycle regulator has been reported in Trypanosoma (Muñoz, et al., 2006), budding yeast (Morris,
et al., 2003), Drosophila (Swan, et al., 2005),
mammalian cells (Spruck, et al., 2003) and a variety of other organisms. However, its function inciliates has remained unknown. Further study of
cdk1 and cks in B. japonicum may provide a
unique system to investigate not only formation of
conjugating pairs but also a wide variety of functions of the cdk–cks complex in ciliates.

ACKNOWLEDGMENT
We thank Prof. Yoshiomi Takagi of Nara
Women's University for helpful suggestion anddiscussion. We are deeply grateful to Prof. Takashi
Miyata and Dr. Naoyuki Iwabe of Kyoto
University for their help. This work was supported
by Grant-in-aids for Scientific research from JSPS
(No. 14580713, No. 18570129) to TH, (No.
17770193, No. 18•714) to MS and by Grant-inaids (2006, 2007) for Young women scientists
from Nara Women’s University to YT.

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