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Original

Paramecium tetraurelia Genes Expressed in an Age-Dependent Manner
Hiroyuki TANABE*, Masaki FUJIIKE and Sachiko YOSHIOKA

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

SUMMARY

INTRODUCTION

In search of the jumyo gene, a putative genethat specifies the clonal life span of Paramecium
tetraurelia, we previously compared gene expression profiles between young wild-type stock 51 andthe short life span jumyo mutant d4-SL4 by thedifferential display technique, and detected four
differentially expressed genes. In this study, assuming that expression of the jumyo gene is continuously altered throughout the clonal life span, we
examined the transcriptional level of the four genes
in the process of clonal aging, but none of thempresented such an alteration. Thus, we again
screened expressed genes of the two strains using
another strategy, cDNA subtraction, and successfully isolated three different genes that show agedependent expression patterns as expected. Of thesethree genes, one was specific to young wild-type
cells and found to code for the 51A surface protein.
The other two were preferentially transcribed in thejumyo mutant, of which one was the α-51D surface
protein gene.

Aging and a limited span of longevity are fundamental realities of organic life and it is clear that
one or more genetic components mediate thesecomplex phenomena (Martin, 2000 and a series ofreviews in the same issue). One promising approach
for identification of these life-controlling factors is
to look for genetic differences that discriminate
long-lived from short-lived species. The jumyo
gene has been reported to be responsible for the
shortened clonal life span of a mutant strain, d4SL4, of the ciliated protozoan Paramecium
tetraurelia (Takagi et al., 1989). In our attempt toelucidate the molecular basis for the Paramecium
life span, we previously screened expressed genesof the short-lived jumyo mutant and its wild-typeparental strain for the prevalence of specific genetranscripts, using the differential display technique(Tanabe and Yoshioka, 2000). Although we isolated four genes that showed differential expression
patterns between the two strains, their involvement
in life span determination was not elucidated.
Normal somatic cells of higher eukaryotes
have a finite number of divisions and telomeres are
considered to be a molecular clock marker for such
limited cellular life span: progressive telomere
shortening at each replicative cycle is associatedwith replicative senescence (Dhaene et al., 2000;
Ishikawa, 2000). Similarly, irreversible changes* Corresponding author
Tel: +81-742-43-1511
Fax: +81-742-43-1445
E-mail: tanabe@nara.kindai.ac.jp

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must be recorded in the expression profile of thejumyo gene as a function of the number of cell fissions, which may underlie the limited longevity of
Paramecium, because the metric of clonal life spanin Paramecium is the number of cell fissions rather
than chronological age (Smith-Sonneborn and
Reed, 1976; Takagi and Yoshida, 1980; Takagi etal., 1987a). A speculative model to explain the fission-counting machinery in the Paramecium lifemaintenance program is as follows: The jumyo genenormally serves to extend the clonal life span.
Hence, its expression is prominent when paramecia
are young but thereafter declines as cell fission
number increases until the termination of expression and consequent clonal extinction. The jumyo
mutant would be defective in this gene. An alternative model is that the jumyo gene operates against
life extension. In this model, its expression, atbackground level in young paramecia, is graduallyenhanced with each cell fission to a threshold, beyond which clonal death results. In this case, a mutational constitutive activation of this gene takes
place in the jumyo mutant.
In this study, we screened genes differentiallyexpressed between young wild-type paramecia andthe jumyo mutant and isolated three genes expressed in correlation with clonal aging. We show
the characterization of these genes and discuss
some of their biological consequences in the cell.

MATERIALS AND METHODS
Strains and cultivation
P. tetraurelia wild-type stock 51 and the jumyo
mutant d4-SL4 (Takagi et al., 1987b) were used andcultivated as done previously (Tanabe and Yoshioka, 2000).
Clonal aging of paramecia
Six cell lines of stock 51 were propagated bydaily single cell isolation (Sonneborn, 1957) until

exautogamous cells senesced and died; the experiments were repeated four times with almost identical results. During cultivation, cells that had freshly
undergone autogamy were clonally aged by dailytransfer of single cells into fresh culture medium,and absence of autogamy was confirmed by inspection of the culture for senescence characteristics such as gradual decrease in growth rate, clonal
extinction at ~ 200 fissions, and changes in cellmorphology and behavior (Takagi, 1988) in parallelwith autogamy check by cell staining (Dippell,1955).
For RNA preparation, a portion of the daily
isolation culture was removed at five stages during
the clonal growth and transferred to mass cultivation to reach ~ 300 cells/ml. Average cell fissions
postautogamy and fission rate (frequency per day ±standard deviation) at each point of the sampling
were: the 1st, 22.1 fissions and 3.8 ± 0.32; the 2nd,60.6 fissions and 3.8 ± 0.29; the 3rd, 95.3 fissions
and 3.2 ± 0.37; the 4th, 139.0 fissions and 2.7 ±
0.77; the 5th, 167.6 fissions and 2.1 ± 1.00.
Construction and screening of subtractive
libraries
Subtracted cDNA libraries were generated
through suppression subtractive hybridization(Diatchenko et al., 1996; Gurskaya et al., 1996), an
improved version of cDNA subtraction. Fig. 1 presents a brief overview of the procedure.
“tester” (the population containing the cDNAs ofinterest) and “driver” (the reference population)
cDNAs were synthesized from their respectivemRNA populations and digested with RsaI. An agesynchronized cell population of young stock 51(about 15 fissions old) and cells of the jumyo mutant (1-12 fissions old) were used as sources ofstarting mRNA. Two different adaptor DNAs wereseparately ligated to the tester cDNAs. After two
rounds of hybridization of the tester with excess ofthe driver, through which tester cDNA species
shared by the driver were subtracted against theJpn. J. Protozool. Vol. 35, No. 2. (2002)

145

Fig. 1. Schematic diagram of the subtractive cloning method.

driver cDNAs, the resulting subtractive tester
cDNAs were selectively amplified by PCR using
primers complementary to the sequences of theadaptors and inserted into the vector pBluescript(Stratagene).
We performed subtractions in both directions:
In the forward subtraction for leaving cDNAs specific to young stock 51, cDNAs derived from young
stock 51 were used as tester and the jumyo mutant
cDNAs served as driver. The reverse subtraction, asecond subtraction done in reverse, was designed toobtain cDNAs present in the mutant (tester) but notin young stock 51 (driver). The resulting subtractive
cDNA pools were greatly enriched for sequences
found exclusively in the tester population, fromwhich two subtractive cDNA libraries were constructed: the forward-subtracted library rich inyoung-paramecia-specific clones and the reversesubtracted library dominated by mutant-specific
ones.

These libraries were then subjected to differential screening to remove cDNA species common
to tester and driver which were still present after
subtraction: 200 cDNA clones randomly pickedfrom each library were dotted (100 ng per spot)
onto two pieces of nylon membrane for duplicate
screening. Each membrane was probed with eitherof the two kinds of the subtracted cDNA pool: onepiece with the forward-subtracted cDNA pool andthe other with the reverse-subtracted one. The filters were then washed under stringent conditions
and exposed to signal detection as previously described (Tanabe and Yoshioka, 2000).
Further screening was carried out by partial
DNA sequencing for removal of redundant clones
and Northern analysis to confirm expression specificity.
Other experimental techniques
Full-length cDNA sequences were obtained by

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RESULTS AND DISCUSSION
Expression of differentiallygenes during clonal aging

Fig. 2. Transcriptional time course of differentiallyexpressed genes during the clonal aging of
Paramecium tetraurelia. A series of culturesof increasing clonal age was prepared fromexautogamous stock 51. One μg of mRNAs
derived from each culture were electrophoresed in a denaturing agarose gel and blotted to anylon membrane. Lanes 1 through 5 containthe RNA samples from the first through thefifth aging stages, respectively (see MATERIALS AND METHODS for details). mRNA of
the jumyo mutant d4-SL4 of 1-12 fissions old(lane 6) was run in parallel with these wildtype samples. Each of the four identical RNA
blots was probed with the WS clone specific toyoung stock 51, either of two cDNA clones,
MS1 and MS2, specific to the jumyo mutant,or a loading control cDNA (from top to bottom). The gene from which the control probe
was generated has proven to be expressed in aconstant degree throughout the Paramecium
life span (data not shown).

rapid amplification of cDNA ends (Chenchik et al.,1996) using cDNA inserts from the subtractive
libraries as templates. Preparation of mRNA,
Northern blotting, and DNA sequence analysis
were done as previously described (Tanabe andYoshioka, 2000). The nucleotide sequence referredto here as MS2 has been deposited in the DDBJ
database with the accession number AB059619.

displayed

As aforementioned, it is our interest whether
the four genes we have cloned by differential display follow the expression mode of the jumyo genein our model: As clonal age of stock 51 increases,
the young-paramecia-specific W gene becomesdepressed, whereas the M1 to M3 genes preferentially expressed in the short-lived mutant become
activated. To address this question, we analyzed thetranscript prevalence of these genes at various
points in the course of clonal aging. mRNA wasprepared at intervals during the daily isolation of
exautogamous stock 51 and subjected to Northernblotting with probes for these genes. However, theresult was inconsistent with the expected one: theW gene maintained the initial level of transcription
throughout the life span and expression of the three
genes specific to the mutant remained negligible
until clonal termination (data not shown). We havedetected only the four clones in a differential display employing 3 x 20 combinations of PCR primer(Tanabe and Yoshioka, 2000). Our target genetranscripts could be displayed with other pairs ofprimer, but a theoretical calculation (Bauer et al.,1993) estimated that screening all species of theParamecium transcripts requires too many primer
combinations for us to deal with.
Cloning of differentially-expressed genes
by subtractive hybridization
Although lack of the transcriptional variation
during clonal aging does not necessarily rule out thefour genes for further study, we preferred isolationof other transcriptional differences between young
paramecia and the jumyo mutant by suppression
subtractive hybridization in light of our model forthe life-span and obtained two subtractive cDNA
libraries: the forward- and reverse-subtracted libraries (Fig. 1). Differential screening revealed thatabout one-fifth of the screened clones produced aJpn. J. Protozool. Vol. 35, No. 2. (2002)

Fig. 3. Physical map of differentially expressedclones. The filled segments indicate the relative location and length of the cDNA fragment of No. 1- 6 clones in the full-length WS(8.2 kb), MS1 (7.6 kb), or MS2 (4.0 kb)
cDNAs. The direction of transcription isarrowed.

markedly and reproducibly stronger hybridization
signal with the corresponding cDNA probe thanwith the other probe in either library (data notshown). The clones that gave the differential signal
indeed represent differentially expressed genes.
We partially sequenced the cDNA insert of thepositive clones to see whether they were unique or
redundant, and accordingly assigned them to several groups representing distinct genes or genefamilies. These positive clones were checked forexpression specificity by probing a Northern blot
containing mRNAs from young stock 51 and thejumyo mutant with cDNA inserts from the individual groups. Every probe hybridized preferentially tothe corresponding mRNA sample, supporting theresult of differential screening (compare lines 1 and6 in Fig. 2 for representative clones). Thus, it was
confirmed that representation of the quantitatively
different transcripts was well retained in our experimental conditions. Overall, five cDNA clones
were isolated as young-wild-type-specific from theforward-subtracted library and eight clones as mutant-specific from the reverse-subtracted library.
Screening for gene expression dependent
on clonal age
We analyzed the differential clones thus ob-

147tained for expression throughout the clonal life spanof stock 51 by Northern blotting as with the previously isolated four clones (Fig. 2). Of the fiveyoung-paramecia-specific clones, two (clone Nos.
1 and 2) had progressively lowered transcription
levels with increasing clonal age to be almost undetectable later in the aging stage (WS in Fig. 2). In
contrast, gene expression continued to increaseduring clonal aging in four clones (clone Nos. 3 - 6)
out of the eight derived from the mutant-specificlibrary (MS1 and MS2 in Fig. 2). As is evident fromthis figure, the transcriptional kinetics in these six
clones is almost superposable on either of the twopatterns of the jumyo expression predicted in ourmodel. As for the remaining clones, little transcriptional change accompanied clonal aging, narrowing
our target genes to the above six clones.
During determination of the complete cDNA
sequences corresponding to the six clones (Fig. 3),
the two young-wild-type-specific sequences, Nos. 1
and 2, appeared separately in the same gene tentatively named WS in Fig. 2. Similarly, three of thefour mutant-specific clones, Nos. 3 - 5, were found
to represent three different regions of one common
gene, MS1 in Fig. 2. The remaining one, No. 6, wasassigned to MS2.
Characterization of target genes
Sequencing the full-length cDNA for the three
genes indicated that WS and MS1 scored perfectmatches with the genes for P. tetraurelia surface
proteins 51A (Nielsen et al., 1991) and α-51D(Brener et al., 1996), respectively, but no meaningful alignment was found for MS2 with available
DNA and protein databases.
The Paramecium surface protein, or immobilization antigen, is a class of proteins with unknown
function and a phenotype resulting from its presence is called serotype (Caron and Meyer, 1989;
Bleyman, 1996). P. tetraurelia stock 51 is able toexpress at least 11 different serotypes including
51A and 51D, and the 51D gene comprises a family

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of isogenes, named α-, γ1-, γ2-, δ-, and ε-51D, inwhich α-51D is expressed in conditions where theD serotype is detected. Serotype expression occurs
in a mutually exclusive manner with only one surface protein present on a cell surface at a time andtransforms to other serotypes in response to environmental changes. Among the P. tetraurelia serotypes, 51A is dominant in standard cultivation(Preer et al., 1987) as done in this study. Our results
indicate that cells of stock 51 present a decreasing
51A expression with a concurrent increasingα51D expression as clonal aging proceeds. Althoughother laboratory has reported serotype transformation as a senescence characteristic of paramecia(Sundararaman and Cummings, 1983), the present
study is the first to identify serotypes showing an
age-associated expression pattern.
Despite its expression associated with Paramecium clonal aging, the 51A serotype is unlikely tocausally participate in the aging process, but is
supposed to be expressed as a consequence of senescence, because P. tetraurelia d48, a mutant
lacking 51A, is healthy (You et al., 1991) andpasses through the normal aging process (Gilly andBlackburn, 1994). The same holds for α-51D. The
dominant serotype in a double mutant unable toexpress the 51A and 51B serotypes appears to be
51D (Preer et al., 1987), making it a plausible ideathat the senescent up-regulation of α-51D is a reciprocal transformation for the down-regulation of
51A. These surface protein genes constituted agreat majority of the subtractive library after thedifferential screening. This high redundancy is accounted for by abundance of mRNAs for surface
proteins. The amount of surface proteins has been
estimated to be about 3.5% of the total protein ofParamecium and their mRNAs are also present inhigh concentration (Preer et al., 1981). In contrast,
genes playing a key role in cellular functions are in
general weakly expressed and therefore poorly
represented in mRNA populations, which is probably the case with the jumyo gene. Although thesubtraction strategy introduces equalization of proportions of particular transcripts during the subtractive process in favor of representation of raretranscripts and the subsequent cloning of theircDNAs (Gurskaya et al., 1996), the quantitative
predominance of the two serotype genes even after
the subtraction suggests that the exceptionally highabundance of the serotype transcripts may adversely affect the equalization step and thereby
make direct cloning of the jumyo gene extremely
difficult. Still, given that there is a causal relationship between the serotype switch and the control ofParamecium longevity, we may be able to isolate
the jumyo gene as the ultimate agent functioning
most upstream the cellular pathway for the serotype
expression by means of genetic and/or biochemical
approaches. Our next targets are regulatory factors
immediately involved in the serotype expression.
At present, we have little information about thefunction of the MS2 gene. In consideration of its
representation in subtractive libraries, this clone ispresumably highly transcribed, similar to serotype
genes. The high abundance of the MS2 transcript isalso supported by its strong Northern blot signal(Fig. 2). Furthermore, MS2 gave rise to multiple
sizes of transcript (Fig. 2). Such a multi-banded
pattern is yielded via at least three mechanisms:
alternative splicing (Smith and Valcarcel, 2000),
use of different transcription initiation sites, and useof different polyadenylation sites. Alternatively,
MS2 may be a member of a multigene family. In
either case, involvement of this polymorphism inthe senescent expression of MS2 is not clear.
Transformational overexpression in wild-type
paramecia and transcriptional repression in thejumyo mutant of this gene should provide insights
into its role and significance in the aging process
and the life-controlling mechanism.
Subtractive cDNA hybridization has been apowerful approach to identify and isolate cDNAs ofdifferentially expressed genes. The incorporation of
PCR into the procedure gave a strong increase inJpn. J. Protozool. Vol. 35, No. 2. (2002)

sensitivity and made it possible to detect differential hybridization signals derived from only a few
transcripts. One potential disadvantage of thistechnique, however, is that the strategy is biased forhigh copy number mRNA and might be inappropriate in experiments in which only a few genes are
expected to vary. The original disproportion in expression level between the group of unusually highabundance, i. e., serotype genes and the newly isolated MS2, and that of rare transcripts is preservedin the Paramecium system, resulting in the escapeof the latter from subtractive cloning. To overcome
this problem, we must extensively examine theequalization of the abundance of cDNAs within thetarget population after subtraction.

ACKNOWLEDGMENTS
We thank Drs. Yoshiomi Takagi and Terue
Harumoto for their valuable suggestions.

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