Jpn. J. Protozool. Vol. 36, No. 2. (2003)

97

Original

Transcriptional Regulation of the MS2 Gene of Paramecium tetraurelia
Hiroyuki TANABE*, and Masato MORI
Laboratory of Biochemistry, Faculty of Agriculture, Kinki University, Nara 631-8505, Japan.

SUMMARY
MS2 of Paramecium tetraurelia is the genethat we have cloned as one expressed at muchhigher levels in a short-lived mutant than in its
parental wild-type stock and have characterized its
expression to be enhanced with increased clonal
age of the wild-type stock. This gene has beenfound to be preferentially expressed also in paramecia undergoing autogamy which is one of thetermination modes of the Paramecium clonal life
span. Here we investigated the transcriptional
mechanism for the MS2 expression in light of its
possible causal relationship to the Paramecium
clonal life span. DNA-protein binding analyses of
the upstream of the MS2 gene identified a stretch of
DNA sequence that interacted specifically withmacronuclear proteins prepared from the MS2expressing mutant. The DNA sequence wasmapped to the 33 bp between – 335 and – 303,
counting from the initiation position of MS2 transcription. A protein, 15,000 in molecular mass, with*Corresponding author
Tel: +81-742-43-1511
Fax: +81-742-43-1445
E-mail: tanabe@nara.kindai.ac.jp
Received: 17 Dec 2002; Accepted: 31 Mar 2003.

a binding ability for this DNA element was purified
to homogeneity from the macronuclear proteins bya chromatography using the specific DNA-protein
interaction. In vitro transcriptional analyses revealed that both the purified protein and its target
DNA domain are essential for increased synthesisof the MS2 transcript. These results showed that the
DNA-protein interaction is required for inductionof the MS2 expression.

INTRODUCTION
For the final purpose of identifying the causal
determinant of the Paramecium clonal life span, thejumyo gene (Takagi et al., 1989), we examineddifferences in gene expression between wild-type
stock 51 and its mutant d4-SL4 with a short clonal
life span, the jumyo mutant, and cloned three genesthat showed expression patterns dependent on
clonal age (Tanabe et al., 2002). Structural analyses(Tanabe et al., 2002) revealed that two of the threewere genes for serotype (Caron and Meyer, 1989;
Bleyman, 1996), 51A and α-51D. The remaining
one was a novel gene named MS2. The MS2 gene,isolated as one expressed predominantly in the
short-lived mutant, was gradually activated in thewild-type paramecia as their clonal age increased(Tanabe et al., 2002). Paramecium tetraurelia
terminates its clonal life span either in clonal death

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MS2 Transcription of P. tetraurelia

or in causing autogamy (Takagi, 1999). In thecourse of another strategy for identification of thejumyo gene, molecular understanding of the
autogamy mechanism, we showed that the MS2
gene became up-regulated in the wild-type stock
through the process of autogamy (Tanabe et al.,2003) The parallel transcriptional patterns of MS2
observed during clonal aging and during autogamysuggest that the two events may share a certain
stage in the genetic program for determination of
the Paramecium life span, in which the gene product of MS2 is functionally participated.
Thus, MS2 is expected to become the firstmolecular basis of the fundamental correlation
between clonal life span and autogamy and theeventual setting of the Paramecium-specific longevity. In support of this notion, a transformation
experiment gave a result suggesting that targeted
repression of the MS2 gene caused a phenotypicallysilencing effect on the occurrence of autogamy(Tanabe et al., 2003), arguing for an intimate
participation of MS2 in the autogamy pathway.
However, the cDNA sequence of MS2 is unlikely
contain some genetic information for translation
products (Tanabe et al., 2003), so it is doubtful
whether MS2 is our final target. Instead, we suppose that the gene product of jumyo serves as thenuclear factor controlling the gene expression thatspecifies the clonal life span by ultimately governing essential cellular functions accompanied withage-dependent phenomenon like the occurrence ofautogamy. If this holds true, elucidation of themechanism for MS2 expression would give someclues for isolation of the jumyo gene. Since expression of Paramecium genes is considered to be
usually regulated at the level of transcription (Preer,1997), our another interest was directed toward theunderlying molecular process for transcriptional
regulation of MS2. Here we dealt with the transcriptional mechanism for the MS2 gene.

MATERIALS AND METHODS
Culture of paramecia
P. tetraurelia wild-type stock 51 and the jumyo
mutant d4-SL4 (Takagi et al., 1987b) were usedand cultivated as done previously (Takagi et al.,1987a; Tanabe et al., 2002).
Preparation of macronuclear proteinsAn age-synchronized cell population of young
stock 51 (about 15 fissions old) and cells of thejumyo mutant were used as sources of protein. Cells
were harvested from a culture of 1,200 - 1,500 cells/
ml and subjected to cellular fractionation for isolation of macronuclei (Shiomi et al., 1980). Nuclear
proteins were extracted from the macronuclear
fraction with the NE-PER nuclear extraction reagent (PIERCE) and dialyzed overnight against theundermentioned binding buffer using a dialysis
membrane with a molecular weight cut-off of
5,000. The dialyzed samples were used as themacronuclear extract in the following experiments.
Cloning of genomic DNA segment
The Paramecium genomic DNA was preparedby a phenol method (Forney et al., 1983). The
upstream segment flanking the MS2 transcribedregion was isolated from the genomic DNA by
inverse PCR (Ochman et al., 1988; Triglia et al.,1988) and the nucleotide sequence of the resulting
897-bp fragment was submitted to the DDBJ database with the accession number AB094989.
Gel retardation assay
Probe DNAs longer than 100 bp were generated by PCR using the above 897-bp DNA as the
template. Shorter probe DNAs were chemicallysynthesized. These probe DNAs were labeled withbiotin using the biotin 3’ end DNA labeling kit(PIERCE).
The DNA-binding experiment (Fried andClothers, 1981) was carried out nonisotopically asJpn. J. Protozool. Vol. 36, No. 2.
1. (2003)

follows: Protein (10 µg in the macronuclear extract
or 10 ng of purified preparation) was preincubatedwith 400 ng of denatured salmon sperm DNA (0.5 1 kb) and, when necessary, 100 ng of unlabeledprobe DNA as a competitor in 19 µl of the binding
buffer (10 mM HEPES-KOH, pH 7.5, 50 mM
NaCl, 8 mM MgCl2, 1 mM dithiothreitol and 10%
glycerol) for 20 min on ice, and then 1 µl of biotinlabeled probe DNA (1 ng) was mixed with thereaction. After an additional 30 min, the mixture
was applied to a 6% polyacrylamide gel (8 cm × 8
cm × 1 mm) containing 0.25 × TBE (22.5 mM Tris,
22.5 mM boric acid and 1 mM EDTA) and electrophoresed at 100 V until bromophenol blue reached
the bottom of the gel. The labeled probe DNA wasthen electroblotted to a nylon membrane in 0.25 ×
TBE at 4 mA/cm2 for 1 h and detected using theLightShift chemiluminescence kit (PIERCE) andan ECL Mini-camera (Amersham).
DNA affinity chromatography
The principle of this procedure is based on a
specific interaction between biotinylated ligand
DNA immobilized on streptavidine-conjugatedagarose beads (PIERCE) and proteins recognizing
the nucleotide sequence of the ligand DNA(Chodosh et al., 1986). After immobilization ofligand, the streptavidine gel, 0.5 ml, was packed
into a column and equilibrated with 2.5 ml of theabove binding buffer. The macronuclear extract, 50
mg in protein, was incubated with 2 mg of denatured salmon sperm DNA (0.5 - 1 kb) for 20 min on
ice and loaded onto the column. Following a thorough washing of the column with 2.5 ml of thebinding buffer containing 0.2 M NaCl, proteinsbound to the ligand DNA were eluted with 1.5 ml of
the binding buffer containing 0.5 M NaCl. The
purified proteins were dialyzed against the binding
buffer as described above for use in the subsequent
experiments.
In vitro run-off transcription99

Two µl of the macronuclear extract was added
singly or in combination with 10 ng of purified
protein to 18 µl of the binding buffer containing 100
ng of template DNA, 1 mM each of ATP, CTP and
GTP, 650 µM of UTP, 350 µM of biotin16-UTP
and 40 units of the ribonuclease inhibitor RNasin.
Transcription was carried out at 25°C for 1 h.
Electrophoresis of the transcript (2 µl) and thesubsequent transfer to nylon membranes followed
our protocol for Northern blotting (Tanabe andYoshioka, 2000). Signals from biotinylated transcripts were detected as described under “Gelretardation assay”.
Other experiments for protein
Protein concentration was determined by themethod of Bradford (1976) with bovine γ-globulin
as the standard protein. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE)
was performed following Laemmli (1970). Gels
after the run were stained with a fluorescent dye(Steinberg et al., 1996; 1997).

RESULTS AND DISCUSSION
Protein-DNA interaction on the upstream of
the MS2 geneIn our preceding study, the MS2 gene wasfound to be transcribed at a low level when wildtype paramecia were young but its transcription wasprominent in the jumyo mutant (Tanabe et al.,2002). This differential gene expression is supposed to result from transcriptional regulation
taking place upstream of this gene: there should
exist a trans-acting nuclear protein factor(s) that
responds to a cis-active DNA element(s) locatedanywhere on the upstream regulatory region, bywhich the gene expression is up- or downregulated.
Assuming that such an intermolecular regulatory interaction occurs preferentially in young wild

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P.MS2
tetraurelia
Transcription
Autogamy-relatedof P. tetraurelia
Genes

Fig. 1. Schematic representation of strategy for location of MS2-specific responsive DNA elements. The 897-bp
macronuclear genomic region (accession No.: AB094989) 5’ to the MS2 transcribed region is presented at the top.
Nucleotides are numbered with the first transcriptional initiation site at position + 1. The direction of transcription isarrowed. Shown below are a set of DNA fragments used as probes in gel retardation assays with their relative sizes andpositions depicted. The filled and open boxes represent probe DNAs testing positive and negative, respectively, in theassays. The nucleotide sequence of Probe 10 is given at the bottom along with the nucleotide positions of its 5’- and 3’ends. The direct repeat within this segment is indicated by arrows facing in the same direction. The nucleotide sequence of Probe 11 is underlined.

type or the jumyo mutant, we searched the macronuclear proteins of the two stocks for DNA-binding
activities specific to the MS2 gene by a gel retardation assay using eleven kinds of DNA fragments
covering the upstream nontranscribed region as theprobe (Fig. 1). First, three fragments, Probes 1 to 3,
spanning – 897 to – 35 relative to the start of thetranscription were examined for interaction withproteins in the macronuclear extract. In the assay
using the proteins of the mutant in which MS2 isactively expressed, Probe 2 (– 595 to – 295) caused
an upward band shift indicating the formation of aprotein-DNA complex (lane 2 in Fig. 2), but themobilities of Probes 1 and 3 were not affected withthe same protein preparation (data not shown). The
shifted DNA band disappeared when an excess of
unlabeled Probe 2 was added as a competitor in thereaction mixture (lane 3), which demonstrated thatthe DNA-protein interaction is sequence-specific.
On the other hand, proteins of young wild type did

not show such an affinity for Probe 2 (lane 1) as
well as for Probes 1 and 3 (not shown). Thus, we
detected a DNA-binding activity toward the MS2
upstream region only in macronuclear proteins of
the jumyo mutant and thereafter exclusively investigated the mutant macronuclear extract.
We next divided the Probe 2 region into threeparts (Probes 4 to 6 in Fig. 1) and assayed them for
response to the mutant macronuclear proteins. In
this screening, the protein sample was found topreferentially retard the mobility of Probe 6, locatedbetween positions – 394 and – 295 (data not shown).
We did further mapping by subdividing the DNA
sequence of Probe 6 into three (Probes 7 to 9 in Fig.
1), and the subsequent deletion analysis of theresulting positive probe, Probe 9 (– 338 to – 295),
finally narrowed down the target sequence to Probe
10, the 33 bp present ahead of position – 303 (notshown).
A close examination of the 33-bp domain

Jpn. J. Protozool. Vol. 36, No. 2.
1. (2003)

Fig. 2. Gel-mobility shift profile of an MS2-specificresponsive DNA element. A gel retardation assay wasdone using either biotin-labeled Probe 2 (see Fig. 1)
and macronuclear proteins either of young wild-type
stock 51 (lane 1) or of the jumyo mutant (lanes 2 and 3)
in the presence (lane 3) or absence (lane 2) of a competitor DNA, unlabeled Probe 2. The filled and open
arrows indicate a free and a protein-bound form of theprobe DNA, respectively.

101

detected a direct repeat of nucleotide sequence,
which is comprised of the consensus sequence 5’TAAACC(C/T)AAA-3’ (Fig. 1). Since manytranscriptional regulator proteins function throughbinding to repeated DNA sequences in their target
regions, the repeat of the decameric sequence islikely to participate in the direct contact with the
mutant macronuclear proteins. To investigate thispossibility, deletion in Probe 10 was assayed forretaining the affinity with the proteins: a partial
deletion in the 3’-half of the repeat made the resultant DNA fragment, Probe 11 (Fig. 1), inactive inthe binding assay (not shown). Consequently, thedirect repeat was identified as the target sequenceon the MS2 gene for the mutant-specific macronuclear factor. A DNA database search did not find thedecameric sequence in any transcriptional regulatory element which has so far been reported.
Purification of a DNA-binding protein specificto the MS2 geneFig. 3. SDS-PAGE profile of the affinity-purified protein. Proteins were electrophoretically size-fractionatedand stained by UV fluorescence. Lanes: 1, molecularweight markers; 2, the macronuclear extract preparedfrom the jumyo mutant; 3, purified preparation after thesecond chromatography. The molecular weights of themarker proteins in lane 1 are given to the left of the lane.

We attempted purification of proteins with thebinding ability for the DNA sequence of Probe 10
by affinity chromatography using the probe DNA
as the ligand. Macronuclear proteins of the mutant
stock were separated by chromatography andproteins retained in the affinity column were eluted
with the running buffer with increased salt concentration.
The profile of the eluted proteins was madevisible by SDS-PAGE: the track in which theproteins were run had one major protein band and a
few faint ones (not shown). To differentiate between all the eluted proteins participating in thespecific contact with the ligand DNA or the eluted
fraction being contaminated with non-specificallybound proteins, this fraction was put through thesecond round of chromatography and the new
eluate was electrophoretically inspected for purity:
the faint multiple band seen after the first purification was lost, while the major band of a protein withmolecular weight of 15,000 appeared again (Fig. 3).

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P.MS2
tetraurelia
Transcription
Autogamy-relatedof P. tetraurelia
Genes

Fig. 4. In vitro transcriptional analyses of the MS2
gene. Run-off transcriptional reactions were carriedout on a template DNA covering the macronuclear
genomic MS2 region from – 338 (lanes 1 and 2) or –295 (lanes 3 and 4) to + 822. The numbering of nucleotide position is the same as used in Fig. 1. The
macronuclear extract of the jumyo mutant was put into
the reactions either alone (lanes 1 and 3) or with theaffinity-purified protein (lanes 2 and 4). The resulting
biotinylated transcripts were subjected to Northernblotting and made visible by chemiluminescence. The
size of the transcript detected here was estimated to beapproximately 0.8 kb by comparison with the migration positions of co-electrophoresed size marker
RNAs (not shown).

Therefore, we ascribed the DNA-binding activity
detected in the binding assay to this protein obtained after the repeated purification. In fact, thesecond purified preparation proved to retard theelectrophoretic mobility of Probe 10 in DNAbinding assays as observed for the mutant macronuclear extract (data not shown), which seems toverify this assignment, but final conclusion must
await structural analyses of this protein.
A titration experiment showed that about 2.5
ng of the purified protein is equivalent in the ability
to retard DNA migration to 10 µg of the mutant
macronuclear proteins, indicating that about fourthousandfold purification on the basis of DNAbinding activity was attained through the chromatography.
Transcriptional activation of the MS2 gene
The molecular interaction between theprotein thus purified and its 33-bp target DNA wasfunctionally characterized by in vitro transcription
reactions on the MS2 gene (Fig. 4). As a positive
control, the mutant macronuclear extract was

incubated alone with a DNA fragment of the MS2
gene beginning 338 bp ahead of and ending 822 bp
past the first transcriptional start site (– 338 to +822) which contains the 33-bp sequence of interest(see Fig. 1), after which synthesis of RNA was
begun. The transcript was run on a denaturing gel,transferred to a membrane, and detected as achemiluminescent signal. Successful RNA synthesis would generate a transcript of about 0.8 kb long.
Imaging of the signal gave a single band at a position corresponding to the expected size (lane 1),
confirming that the nuclear extract can elongate
nascent RNA chains while incorporating ribonucleoside-triphosphates including a biotin-labeled
one. When the affinity-purified protein was added
to the reaction, the transcription was found to be
stimulated several times (lane 2). Since the input ofthe purified protein, 10 ng, is about four times theequivalent of the nuclear extract used here in termsof DNA-binding ability (see above), these two data
together are represented as the synthesis of the MS2transcript in proportion to the DNA-binding activity, which is best explained by assigning the transcriptional increase to the binding of the protein to
its target element. Actually, in the transcription
experiments using a template DNA devoid of the
33-bp domain (the region – 295 to + 822), therelative intensity of signals from the resulting
transcripts was below detectable levels with (lane4) or without (lane 3) the protein.
Taken together, we concluded that the protein
purified in this study serves as a trans-acting factor
for positive regulation of the MS2 transcription
through the specific interaction with the – 335 to –303 domain, and thereby induces the MS2 expression in the jumyo mutant. Extended investigationsof the DNA-protein interaction in the MS2 expression as well as structural characterization of thetrans-acting factor should elucidate the underlying
regulatory mechanism and its role in setting theParamecium clonal life span.

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Genes

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