Jpn. J. Protozool. Vol. 36, No. 1. (2003)

1

Original
Isolation and Characterization of Genes Showing Altered Expression
Specific to Autogamy in Paramecium tetraurelia
Hiroyuki TANABE*, Masaki FUJIIKE and Sachiko YOSHIOKA

Laboratory of Biochemistry, Faculty of Agriculture, Kinki University, Nara 631-8505,
Japan.

SUMMARY

INTRODUCTION

Autogamy is a sexual process of meiosis andself-fertilization which occurs in unpaired Paramecium cells and is considered to be coupled to theParamecium clonal life span. In order to identify
gene expression which may underlie the initiationof autogamy, we screened for genes differentiallyexpressed before and after the onset of autogamy byusing a subtraction strategy and obtained two species of difference genes. Both genes were found tobe the ones increasingly expressed with clonal age
that we have cloned previously. One was repressed
upon commitment to autogamy and identified to be
the α-51D surface protein gene. The other, progressively activated while paramecia traversed theautogamy pathway, was identical to the gene named
MS2. Although we failed to structurally characterize the MS2 gene, it was suggested that continuous
delivery to paramecia of double stranded RNA
corresponding to a part of this gene impaired normal development of autogamy, implying direct
involvement of MS2 in the process of autogamy.

The ciliated protozoan Paramecium tetraurelia
undergoes two forms of sexual reproduction, bothof which include meiosis, fertilization, and macronuclear development (Sonneborn, 1974). One,conjugation, develops from surface interaction between sexually competent cells of complementarymating types. The other, autogamy, is an intracellularly initiated process of self-fertilization in single
cells which is triggered by food deprivation. Termination of the Paramecium clonal life span can be
defined by either clonal death or entry into thesesexual pathways. Paramecia count a definite number of cell fissions to traverse the period from theclonal origin to clonal death. Likewise, undergoing
sexual reproduction requires a certain number offissions after the preceding fertilization, the duration of which is called the immaturity period. Conjugation and autogamy have their own period for
immaturity, sexual immaturity and autogamy immaturity, respectively, and thereafter paramecia
become mature, that is, capable of entering eachsexual pathway.
A positive correlation has been establishedbetween the ciliate clonal life span and the length of
the sexual immaturity period (Smith-Sonneborn,1981). Clonal age of parent cells has an adverse
effect on the duration of sexual immaturity (Siegel,1961) and of autogamy immaturity (Iizima et al.,*Corresponding author
Tel: +81-742-43-1511
Fax: +81-742-43-1445
E-mail: tanabe@nara.kindai.ac.jp
Received 29 Oct 2002; Accepted 24 Dec 2002

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P. tetraurelia Autogamy-related Genes

1997) in their progeny. Autogamy immaturity issimilarly affected by parental cultural age as well(Ishikawa et al., 1998). In the jumyo mutant of P.
tetraurelia, which has an extremely short life span,
the immature period for autogamy is also shortened(Takagi et al., 1987; 1989). These findings allow
the speculation that the mechanism for setting theclonal life span and that for initiating sexual reproduction are tightly linked (Takagi, 1999).
The molecular aspects of the control of theParamecium sexual reactivity have been elucidated
to some extent. Immaturin, a small cytoplasmic
protein, was identified as the factor responsible forsexual immaturity (Miwa et al., 1975; Haga and
Hiwatashi, 1981; Miwa, 1984). There seems toexist the autogamy version of immaturin. Transplantation of macronuclear material or the entire
macronuclei between autogamy-immature and mature cells significantly influences the occurrence ofautogamy in P. tetraurelia (Kosciuszko and Koizumi, 1983; Mikami and Koizumi, 1983). Together
with the cell-cycle-stage-dependence of commitment to autogamy (Berger, 1986), these experiments indicate that a nuclear factor(s) is directly
involved in the regulation of the autogamy pathway
and hence particular alterations in gene expression
must be associated with this sexual event. We havecompared gene expression profiles between the
short-lived jumyo mutant and its wild-type parent
stock and isolated differentially expressed genes forthe final purpose of identification of the causal determinant of the Paramecium clonal life span
(Tanabe and Yoshioka, 2000; Tanabe et al., 2002).
In view of the above argument, investigation of
autogamy-specific gene expression would be another important approach to our goal. This paper
describes an initial step in the second approach.

P. tetraurelia wild-type stock 51 and the jumyo
mutant d4-SL4 (Takagi et al., 1987) were thestrains used in this study. Daily isolation line
culture (Sonneborn, 1954) was developed fromexautogamonts of the wild-type stock as a sourceof cells mature for autogamy. To obtain preautogamous, autogamy-committed, and postautogamous cells at the age of about 35 fissions,cultures derived from the isolation culture wereallowed to reach 400-500, 1,200-1,500, and 4,0005,000 cells/ml, respectively. The clonal age andpopulation density for each autogamy stage were
determined on the basis of kinetic experiments byBerger and Rahemtullah (1990). We detectedoccurrence of autogamy by cell staining (Dippell,1955) and observed that the percentages of
autogamonts in each culture phase were 0, about
5%, and about 80%, respectively. The cell densityof 1,200-1,500 cells/ml has been estimated to bethat at the median time of initial commitment toautogamy for 35-fission old paramecia (Berger,
1986; Berger and Rahemtullah, 1990). The
committed cells undergo autogamy after thefollowing cell fission, the “critical fission”(Mikami and Koizumi, 1983). Actually, thepercentage of autogamy was raised to about 60%
after one fission, i.e., at about 3,000 cells/ml,
indicating that more than half of paramecia at
1,200-1,500 cells/ml were in the state of autogamy
commitment under the conditions used here.
Therefore, we considered paramecia at each of theabove three culture phases to be satisfactory in thepresent study as a cell material for the corresponding autogamy stage. Liquid medium and othercultivation conditions were mentioned before(Tanabe and Yoshioka, 2000).
cDNA subtraction

MATERIALS AND METHODS
Culture of paramecia

Construction and screening of subtractive
cDNA libraries were carried out according to our
protocol (Tanabe et al., 2002). In the present
experiment, mRNAs prepared from matured wild-Jpn. J. Protozool. Vol. 36, No. 1. (2003)

3

type cells in a preautogamous state and thosecommitted to autogamy were used as the starting
materials. Two subtraction experiments were
designed to enrich genes expressed preferentiallyeither in the preautogamous or in the autogamycommitted cells: the forward subtraction to recoverpreautogamy-specific cDNAs (tester, preautogamous cell population; driver, autogamycommitted cell population) and the reverse subtraction to leave cDNAs specific to autogamy
commitment (tester, autogamy-committed cell
population; driver, preautogamous cell population).

cDNA of MS2 as probes. Two Paramecium cells
were transferred into 2 ml of the RNA-feeding
culture thus prepared and their phenotypic
response was observed after a 5-day successive
cultivation at 25°C.

Plasmid DNA and host bacteria for dsRNA
expression

RESULTS AND DISCUSSION

DNA fragments were generated by PCR
amplification (Tanabe and Yoshioka, 2000) using
the entire cDNA sequence of MS2 (DDBJ
accession no. AB059619) as the template. The
primers used here were tagged with the T7
promoter sequences to allow synthesis of double
stranded RNA (dsRNA) from the resulting PCR
products by T7 RNA polymerase (Tsuboi et al.,2002). The amplification products (see Fig. 2)
were inserted into the pGEM-T cloning vector(Promega) and introduced into Escherichia coli
HT115 (DE3) (Takiff et al., 1989; Dasgupta et al.,1998) used as a food bacterium for delivering thedsRNA to Paramecium cells.
Feeding of bacterially expressed dsRNA toparamecia
The above E. coli strain harboring plasmid
was cultivated in the Paramecium liquid medium
at 37° C to an optical density at 600 nm of
approximately 0.4. To induce T7 RNA polymerase, isopropyl-β-D-thiogalactopyranoside wasadded to 0.4 mM and further cultivation was donefor 2 h. Synthesis of the dsRNA was confirmed by
Northern blotting using a sense and an antisense

Other experiments
Preparation of mRNA, Northern blotting,
DNA sequence analysis and rapid amplification of
cDNA ends were described before (Tanabe andYoshioka, 2000; Tanabe et al., 2002).

Subtractive cloning of genes expressed differentially during autogamy
In the conviction that underlying transcriptional changes precede the prosecution of
autogamy, we performed isolation of genes up- or
down-regulated on conversion from preautogamous to autogamy-committed stages by means of
cDNA subtraction. Following a series of screening
steps, one each of cDNA clone was finally isolated
from the forward- and the reverse subtractions.
The resulting preautogamy- and autogamy- specific clones were tentatively designated as NS and
AS, respectively. Fig. 1 depicts their transcription
patterns during the development of autogamy. The
expression of the preautogamy-specific NS clone
declined abruptly to a basal level when cells werecommitted to autogamy. On the contrary, the AS
clone from autogamy-committed stage is positively regulated over the course of the autogamy
process. Its transcriptional level remained elevateduntil the completion of autogamy.
Identification of autogamy-specific genes
A search of the DDBJ database revealed thatthe two clones were identical to the α-51D geneand MS2. These genes are two of the three genes

4

P. tetraurelia Autogamy-related Genes

Fig. 1. Northern blot analysis of autogamydependent gene expression in Paramecium
tetraurelia. mRNA samples (1 µg each) derived
from preautogamous (lane 1), autogamy-committed(lane 2), and postautogamous (lane 3) mature cells ofstock 51 were separated by electrophoresis andtransferred onto nylon membranes to prepare a triplicate blot. Each blot was hybridized with a cDNA
probe for NS, AS, or control for mRNA load (fromtop to bottom). The control gene has proven to betranscribed independent of the autogamy stage in apreliminary experiment.

isolated in our preceding study, where they
showed much higher expression in the short-lived
jumyo mutant than in its wild-type parental strain
as well as increasing expression during clonal aging (Tanabe et al., 2002): We found the nucleotide
sequence of the NS clone in the gene that encodes
the α-51D surface protein specifying the D serotype of P. tetraurelia (Brener et al., 1996). The
unique multiply banded profile of the AS transcripts (Fig. 1) is reminiscent of the MS2 genewith the accession number AB059619. Indeed,both DNA sequences were aligned with a perfecthomology.
Since aged paramecia are prone to undergo
autogamy even when they are well fed and thesame is the case for the jumyo mutant (Takagi etal., 1987), it was conceivable that the α-51D gene
up-regulated in these cells is similarly regulated in

the autogamy process. However, Fig. 1 shows aresult to the contrary. Although the observed
autogamy-dependent repression of this serotype
gene is difficult to reconcile with its agingdependent activation, one possible explanation for
the two opposite expression pattern is that the aging pathway and the autogamy pathway involve
their respective independent steps, to each ofwhich α-51D contributes in a different manner. In
any case, it is likely that this cellular surface protein receives adverse environmental stimuli asfood restriction and regulates the subsequent intracellular transmission of the external signals. This
notion is supported by a structural analysis(Nielsen et al., 1991) indicating that Paramecium
surface proteins are potentially capable of intermolecular protein interactions.
While, the MS2 gene with little known function is worth noting. Its transcription mode in thecourse of autogamy (AS in Fig. 1) is parallel withthat during clonal aging (Tanabe et al., 2002). This
close transcriptional association between the two
events could reflect their intimate interrelation(Takagi, 1999) as introduced above, in which theMS2 gene product may play an important role.
Thus, our interest was focused on the relationshipbetween the MS2 gene and the Paramecium
autogamy.
Structural analysis of MS2
As shown in Fig. 1, the MS2 gene generates
three distinct transcripts, for which several interpretations have been offered: diversity in the splicing point, in the transcription starting point or in
the polyadenylation point (Tanabe et al., 2002). To
discriminate between those possibilities, we analyzed 30 independent cDNA clones of this geneisolated by rapid amplification of cDNA ends.
They were sorted into three groups according tosize: about 1.8, 2.4 and 4.0 kb, which were close to
the size of the MS2 transcripts estimated fromtheir Northern blot. Sequencing of representativeJpn. J. Protozool. Vol. 36, No. 1. (2003)

Fig. 2. Maps of MS2 transgene sequences used in
dsRNA-feeding experiments. Two DNA segmentsof MS2, the 5’ half of cDNA (the upper open box)
and the full-length cDNA (the lower open box),
carrying tags of the T7 promoter (solid boxes) onboth ends were cloned into pGEM-T and the resulting constructs were named pMS2- and pMS2+,
respectively. Nucleotide positions are given relative
to the first transcriptional start (+1) of MS2.

clones belonging to each group revealed that the
1.8-kb and 2.4-kb clones are shorter isoforms representing the 3’ portion of the 4.0-kb clone, thefull-length cDNA (4,044 bp), and have the startsites located 2,255 bp (+2,255) and 1,606 bp(+1,606) downstream the 5’ terminus of the fulllength clone. Therefore, we concluded that thesedifferentially transcribed variants of MS2 resulted
from use of alternative transcriptional start.
We next searched the 4,044-bp cDNA sequence for protein coding frames. In Paramecium,
TAA and TAG specify glutamine and TGA alone
is used as a translational stop (Caron and Meyer,
1985; Preer et al., 1985). The three possible reading frames, however, all contained scattered TGA(stop) codons, making it difficult to define thetranslated domain. So, we applied to this study themethod for protein frame determination frompurine/pyrimidine information (Shepherd, 1981):(1) Codon usage in all organism is restricted suchthat the ratio of RNY to YNR codons, where R is
purine, N any base and Y a pyrimidine, is > 2 fortrue reading frames, and < 1 for noncoding frames.
(2) In organisms having A+T-rich genomes, coding regions have a much lower A+T content thannoncoding regions and are characterized by a 3-bp
periodicity in A+T richness, i.e., the third base ofeach codon shows the strong bias for A and T.
Among numerous ATG-TGA frames present in the5

MS2 cDNA sequence, only the 192 bp starting at
position +707 counting from the third transcriptional initiation point (+2,255 relative to the 5’ terminus) followed the first rule: its RNY/YNR ratiois 2.1, but this DNA segment did not satisfy thesecond criterion: it has 65% A+T at the third position versus 70% A+T in the entire sequence, displaying no such periodicity in A+T content. In addition, the 192-bp frame shares only about one
tenth the shortest transcribed portion (1.8 kb) of
MS2, which seems to be unlikely for a translation
moiety, and the amino acid sequence of 63 residues deduced from its nucleotide sequence showed
little homology with other known proteins. Together with these data, it is doubtful whether the
DNA sequence of MS2 actually encodes a certaintranslation product(s).
Functional characterization of MS2
From the above results, we did not obtain any
information about the role of MS2 in the autogamy
event, so we investigated phenotypic influence ofrepression of this cryptic gene on autogamy bymeans of RNA interference (RNAi). RNAi is aposttranscriptional gene-silencing phenomenon inwhich exogenous dsRNA triggers degradation ofhomologous cellular mRNA of target gene,
thereby the target gene is repressed (Brantl, 2002;
Hannon, 2002), and has been proven to work for
Paramecium as well (Kung et al., 2000). In thisstudy, we employed bacterially mediated RNAi
(Timmons and Fire, 1998; Timmons et al., 2002;
Galvani and Sperling, 2002): Transformation was
performed by feeding autogamy-competent paramecia with food bacteria producing high levels of
dsRNA derived from MS2 transgenes.
We first assessed phenotypic changes withrespect to autogamy in matured wild-type paramecia (about 50 fissions old) that were fed bacteria
expressing dsRNA from pMS2- lacking the 3’ halfof MS2 or pMS2+ containing the entire MS2 (Fig.
2). Previous studies demonstrated that gene silenc-

6

P. tetraurelia Autogamy-related Genes

Table 1. Phenotypic characterization of paramecia transformed with MS2 transgenes*
Host strain
Plasmid

Wild-typed4-SL4

pGEM-T

58.5% (20)

90.2% (18)pMS2-

51.1% (20)

91.7% (14)pMS2+

62.8% (20)

92.0% (19)*The dsRNA-feeding experiment was done with 20
independent cultures for each combination of hoststrain and plasmid. Results were expressed as the fraction of autogamonts in the cell population inspected for
macronuclear fragmentation using Dippell’s stain after
a 5-day consecutive cultivation. The percentages were
the average of the results of the 20 cultures which did
not differ by more than 4%. The values in parenthesis
indicate the number of the cultures that survived the 5day cultivation out of the 20 cultures.

ing takes place by transforming paramecia withtransgenes lacking the 3’ noncoding region of target genes (Ruiz et al., 1998), but the effect can not
be obtained with transgenes bearing the flanking
regulatory region (Galvani and Sperling, 2001). If
MS2 intimately participates in the autogamy pathway, paramecia transformed with pMS2- shouldpresent a silenced phenotype connected withautogamy and the interference phenomenon should
not appear with pMS2+. Data in Table 1 seem toagree with this assumption: the wild-type cells that
fed on bacteria harboring pMS2- displayed a weak
but distinct resistance to undergoing autogamy incomparison to feeding of pMS2+ as well as thecontrol plasmid pGEM-T.
We then carried out feeding of bacteria engineered to express the MS2 constructs to the jumyo
mutant d4-SL4. In this case, clonal descendants
that were exposed to pMS2- showed a reduction in
viability with a significant difference from that inreference experiments using pGEM-T and pMS2+(14 cultures for pMS2- versus 18 cultures for
pGEM-T and 19 cultures for pMS2+), while liability to autogamy was unaffected as for surviving

cells which were supposed to successfully escape
the transformation by pMS2- (91.7% versus 90.2%
and 92.0%). The jumyo mutant terminates its
clonal life span either in early death or in preferred
autogamy (Takagi et al., 1987; Takagi et al.,1989), i.e., escapes clonal death by causing
autogamy. Consequently, genetic interference of
the autogamy process should cause a lethal effecton the mutant cells in the vegetative growth. The
lowered viability of the mutant for pMS2- can be
explained straightforwardly by assuming that repression of MS2 adversely affected the occurrenceof autogamy in the mutant cells, which resulted intheir clonal death.
In this study, we obtained presumptive evidence for functional participation of MS2 in the
Paramecium autogamy, but the susceptibility ofparamecia to silencing transgene observed in our
feeding experiments was significantly lower thanthe case of other Paramecium genes so far reported (Galvani and Sperling, 2002), so that assignment of the inhibitory effect of pMS2- on
autogamy to the RNAi phenomenon is a matter of
debate. For certain target genes and experimental
regime, higher RNAi efficiency has resulted fromtransgenes directly injected into host cells thanfrom those delivered by feeding (Timmons andFire, 1998; Timmons et al., 2002). Delivery of theMS2 transgene by microinjection may trigger
more distinct inhibitory effect and make the assignment conclusive. Besides, fine mapping of theMS2 sequence required for the phenotypic change
should help the molecular understanding of therole of MS2 in the Paramecium autogamy pathway.

ACKNOWLEDGMENTS
We thank Dr. Yoshiomi Takagi for valuablesuggestions.

Jpn. J. Protozool. Vol. 36, No. 1. (2003)

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