I) Preparation of parasites
P. falciparum 3D7 parasites were cultured either in a candle jar
(XPFn, XPF2n) or in the mixed gas consisting of 3% oxygen, 5% carbon dioxide
and 92% nitrogen using blood type A erythrocytes and frozen human plasma.
Blood samples were collected from the Indonesian patients with P.vavax
who were diagnosed by Giemsa staining of the blood smear and gave the
written informed consent. Heparinized whole blood was filtrated through
Plasmodipur filter (Eurodiagnostica) to remove white blood cells. Parasites
were homogenized using the Polytron Homogenizer, frozen in liquid nitrogen
and sent to Japan with dry ice.
Plasmodium yoelii and Plasmodium berghei
P. yoelii 17XL (XPYw), P. yoelii 17XNL (XPYs) and P. berghei ANKA
were propagated in vivo in five-week old female ICR mice. Blood was
filtrated through Plamsodipur filter to remove white blood cells.
The RH strain of Toxoplasma gondii tachyzoites (1x107 parasites)
were infected to Vero (T25) cells and incubated in EMEM medium supplemented
with 8% fetal bovine serum for 72 hours in vitro. Infected cells were
collected with a scraper and passed through 27G needle for three times and
purified by filtration through Millex SV filters (pore size 5 um,
Millipore). Cells were collected by centrifugation at 2,000 rpm for 10 min
and washed in EMEM medium without FBS. Parasites were suspended at 1x107/ml
in EMEM medium without FBS. Five-week old ICR female mice were infected
with 1 ml parasites intraperitoneally.. Three days after infection, mice
were sacrificed by cervical dislocation. Parasites were collected by
washing the peritoneal cavity with 5 ml EMEM, passed through 5 um filter,
washed in PBS and suspended at 2-3x109 parasite/ml.
20 ml TRI REGENT (Sigma) was added to 1 ml parasite solution, mixed by
pipetting, frozen in liquid nitrogen and kept at -80C until used.
C. parvum HNJ-1 strain (genotype 2) was originally isolated from a
Japanese immunocompetent patient with diarrhea in 1989 and has been
maintained by subinoculation in SCID (severe combined immunodeficiency)
Five-week old female SCID mice were infected by oral inoculation of
105 oocysts. Feces were collected in water under the cage and oocysts were
purified by sugar floatation followed by diethyl ether method. Oocysts were
incubated in 0.1N HCl at 37oC for 1 hour. After three washes with MEM they
were incubated in MEM supplemented with 0.1% deoxycholate at 37oC for 30
min. Excysted sporozoites were homogenized in Trizol (Invitrogen) using
Polytron homogenizer and RNAs were isolated.
II) Isolation of RNA.
Cytoplasmic RNA and Poly A+ RNA were isolated using standard methods.
Oligo-dT cellulose was obtained from Collaborative Biomedical Products
Oligo-capping was performed as described (Maruyama and Sugano,
1994), with some modifications. In brief, 10 to 50 ug of polyA+ RNA
was treated with 1.2 units of bacterial alkaline phosphatase (BAP; TaKaRa)
in 100ul of 100mM Tris-HCl (pH 8.0), 5mM 2-mercaptoethanol with 100 units
of RNasin (Promega) at 37oC for 60 min. After extraction with phenol:chloroform
(1:1) and ethanol precipitation, the polyA+ RNA was treated with 20 units
of tobacco acid pyrophosphatase (TAP) in 100ul of 50mM sodium acetate
(pH 5.5), 1mM EDTA, 5mM 2-mercaptoethanol with 100 units of RNasin at
37oC for 60 min. After phenol:chloroform extraction and ethanol precipitation,
2 to 4ug of the BAP-TAP treated polyA+ RNA were ligated with 0.4ug of
5'-oligo (KM-02; 5'-AGC AUC GAG UCG GCC UUG UUG GCC UAC UGG-3') using
250 units of RNA ligase (TaKaRa) in 100ul of 50mM Tris-HCl (pH7.5), 5mM
MgCl2, 5mM 2-mercaptoethanol, 0.5mM ATP, 25% PEG8000 with 100 units of
RNasin at 20oC for 3 hours.
IV) cDNA synthesis.
After unligated 5'-oligo was removed, cDNA was synthesized with RNaseH
free reverse-transcriptase (Superscript II, Gibco BRL). Ten picomoles
of dT adapter-primer (5'-GCG GCT GAA GAC GGC CTA TGT GGC CTT TTT TTT TTT
TTT TTT-3') was used in 50ul with 2 to 4ug of oligo-capped polyA+ RNA.
The reaction conditions were as recommended by the supplier and reactions
were incubated at 42oC for 3 hours.
V) cDNA amplification.
After first-strand cDNA synthesis, RNA was degraded in 15mM NaOH by incubating
at 65oC for 1 hour. The cDNA which is made from 1ug of "Oligo-capped"
polyA+ RNA was amplified in a volume of 100ul using an XL PCR kit (Perkin-Elmer)
with 16pmol of 5' (5'-AGC ATC GAG TCG GCC TTG TTG-3') and 3' (5'-GCG GCT
GAA GAC GGC CTA TGT-3') PCR primers. For dT-adapter primer primed cDNA,
amplification were carried out by 15 cycles of PCR reactions at 94oC for
1 min, 58oC for 1 min, and 72oC for 10 min. PCR products were extracted
with phenol:chloroform (1:1) once, ethanol precipitated and digested with
SfiI. SfiI-digested PCR products were separated by agarose
gel electrophoresis and products longer than 1 kb were isolated and cloned
into DraIII-digested pME18S-FL3.
In this way, we could clone the cDNA into the vector in an orientation-defined
Plasmid DNA was isolated using PI-100 and PI-200 auto-plasmid-isolators
were determined by the dideoxy termination method using an AutoCycle sequencing
kit (Pharmacia) and a reaction robot R. O. B. DNA processor (Pharmacia)
or a BigDye sequencing kit (ABI). The sequence was read using an ALF DNA
(Pharmacia) and an ABI 377XL (ABI) auto-sequencer.
VII) Sequence similarity test.
Sequence similarity of cDNA was tested against GenBank non-redundant nucleotide
library (Release 98) using the BLASTN or FASTA program.
VIII) Database construction.