Targeted Reduction of Nucleoside Triphosphate Hydrolase by
Antisense RNA Inhibits Toxoplasma gondii Proliferation*
Valerian
Nakaar,
Benjamin U.
Samuel,
Emily O.
Ngo, and
Keith A.
Joiner
From the Department of Internal Medicine, Yale University School of
Medicine, New Haven, Connecticut 06520-8022
 |
ABSTRACT |
Nucleoside triphosphate hydrolase (NTPase) is a
very abundant protein secreted by the obligate intracellular parasite
Toxoplasma gondii shortly after invasion of the host cell.
When activated by dithiols, NTPase is one of the most potent apyrases
known to date, but its physiological function remains unknown. The
genes encoding NTPase have been cloned (Bermudes, D., Peck, K. R.,
Afifi-Afifi, M., Beckers, C. J. M., and Joiner, K. A. (1994) J. Biol. Chem. 269, 29252-29260). We have
recently shown that the enzyme is tightly controlled within the
vacuolar space and may influence parasite exit from the host cell
(Silverman, J. A., Qi, H., Riehl, A., Beckers, C., Nakaar, V., and
Joiner, K. A (1998) J. Biol. Chem. 273, 12352-12359).
In the present study, we have generated an antisense NTP RNA construct
in which the 3'-untranslated region is replaced by a hammerhead
ribozyme. The constitutive synthesis of the chimeric antisense
RNA-ribozyme construct in parasites that were stably transfected with
this construct resulted in a dramatic reduction in the steady-state
levels of NTPase. This inhibition was accompanied by a decrease in the
capacity of the parasites to replicate. The reduction in parasite
proliferation was due to a specific effect of antisense NTP RNA, since
a drastic inhibition of hypoxanthine-xanthine-guanine phosphoribosyl
transferase (HXGPRT) expression by a chimeric antisense HXGPRT
RNA-ribozyme construct did not alter NTPase expression nor compromise
parasite replication. These data implicate NTPase in an essential
parasite function and suggest that NTPase may have more than one
function in vivo. These results also establish that it is
possible to study gene function in apicomplexan parasites using
antisense RNA coupled to ribozymes.
 |
INTRODUCTION |
Apicomplexan parasites (e.g. Toxoplasma,
Plasmodium, Cryptosporidium, and
Eimeria) are prevalent worldwide and cause significant diseases in
humans and animals. In particular, the obligate intracellular parasite
Toxoplasma gondii is an opportunistic infection associated with AIDS and congenital neurological birth defects (1). Treatment regimens for this infection are not always effective, and this has
mandated the search for novel drug targets.
As reported previously, we and others have identified the gene encoding
nucleoside triphosphate
(NTPase),1 a very abundant
protein secreted by T. gondii shortly after invasion of host
cells (2-5). The 63-kDa NTPase isoforms are encoded by two genes,
NTP1 and NTP3, which share more than 97%
sequence identity (2). When activated in vitro by dithiols,
NTPase is one of the most potent apyrases known to date, capable of
sequentially degrading ATP to ADP and AMP (4). Because T. gondii is a purine auxotroph, it is thought that NTPase may
participate in purine salvage (2, 6, 7). Recently we have shown that
the enzyme may facilitate parasite exit from infected host cells (8). However, the biological function of NTPase in vivo is yet to
be defined.
It is possible to generate null mutants by gene knock out in the
apicomplexan parasites (9-11). This strategy enables the study of gene
function that can lead to the identification of potential drug targets.
However, the presence in the genome of multiple copies of a gene (such
as NTP) combined with low frequency of homologous
recombination in T. gondii, hamper gene targeting efforts in
these organisms. Moreover, it is not feasible to investigate the
biological function of essential genes using this approach because of
the lethality of the phenotype. To circumvent these difficulties, we
sought to develop an antisense strategy to inhibit gene expression in
T. gondii.
Antisense RNA has been shown to regulate gene expression in bacteria
(12), Dictyostelium (13), Leishmania (14),
Drosophila (15), Xenopus oocytes (16), mammalian
cells, (17) and plants (18-20) but has not yet been demonstrated in
the apicomplexan parasites. This strategy is at present still by
trial and error. This may be partly ascribed to the finding that in
eukaryotes, 3'-end formation of mRNA promotes the export of the
mature transcript to the cytoplasm (21). Since antisense-induced
mRNA degradation in eukaryotes occurs in the nucleus (22), the use
of a polyadenylated RNA may reduce the overall efficiency of antisense
RNA. Eckner et al. (21) have shown that substituting the
normal polyadenylation signal with cis-acting ribozymes
leads to the nuclear retention of the product RNAs by generating
export-deficient transcripts (21). Ribozymes are catalytic RNA
molecules that are active in vitro, both in cell-free
systems and in living cells (23-26) and are currently being developed
for gene therapy applications as inhibitors of gene expression and
viral replication (27-28).
In this report we show that the expression of NTPase can be
substantially inhibited by stably introducing into the parasite genome
an antisense gene in which its 3' end is modified by a hammerhead
ribozyme. The attenuation of NTPase activity inhibits parasite
proliferation, suggesting a role for NTPase in parasite metabolism. The
inhibition of parasite replication was due to specific reduction in
NTPase by antisense RNA, since a drastic reduction in
hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT)
expression did not alter NTPase expression nor compromise parasite
replication. The chimeric antisense RNA-ribozyme strategy should be
useful for the study of gene function in other protozoan parasites.
 |
MATERIALS AND METHODS |
Parasite Cultures, Transfection, and Parasite
Replication--
RH-strain of T. gondii parasites were
cultivated in Vero cells or in primary human foreskin fibroblasts
(HFF), and the tachyzoites were transfected with plasmid constructs
p
NTP-RZ, pASHXRZ, or p5'NTP-RZ (see Fig. 1) subcloned into a vector
containing the dihydrofolate reductase-thymidylate-synthase
(DHFR-TS) gene, which confers pyrimethamine resistance for
selection (29). Stable transformants were selected in 1 µM pyrimethamine, and individual clones were isolated.
Confluent cultures of HFF cells in 10-cm plates or 25-cm2
T-flasks were inoculated with 106 freshly lysed-out
parasite tachyzoites and incubated at 37 °C for 4 h, and the
cultures were replaced with fresh medium so as to remove parasites that
had not yet invaded. The doubling of intracellular parasites was
followed by counting the number of tachyzoites per parasitophorous
vacuole at the various times (at least 50 vacuoles were scored at each
time point). The number of parasite divisions since infection was
determined using log2 (parasite number) formula (30).
Statistical significance was assessed by measures of analysis of
variance and two-tailed t test.
Enzyme and Uracil Incorporation Assays--
NTPase enzymatic
activity was done as described previously using equivalent amounts of
parasite extracts (2). Briefly, 1-10 µg of cell lysate was incubated
with [2,8-3H]ATP (Amersham Pharmacia Biotech) in the
presence of 100 mM HEPES-KOH, pH 7.2, 30 mM
magnesium acetate, 10 mM ATP, 0.1 mg/ml soybean trypsin
inhibitor, and 1 mM dithiothreitol at 37 °C for 10 min. The reaction products were separated by TLC, spots corresponding to the
nucleobase and 5' products were excised, and the radioactivity was
counted under liquid scintillation. HXGPRT activity and parasite replication were assayed by measuring the incorporation of
[8-3H]xanthine (Moravek Biochemicals) and
[5,6-3H]uracil (Amersham Pharmacia Biotech),
respectively. 2-5 × 105 parasites/well were used to
infect monolayers of HFF in a 24-well plate at 37 °C. Unattached
parasites were removed 4 h later, and after 24 h
post-infection, 1 µCi of radiolabel was added to each well.
Incubation was continued for 2-6 h before the monolayers were fixed
with trichloroacetic acid, rinsed, and counted as described (31,
32).
Northern and Western Blot Analyses--
Total RNAs were isolated
from parasites with TRIzol reagent (Life Technologies, Inc.), and
Northern blot analyses were performed with 10 µg of total RNA using
gel-purified NTP cDNA probe (corresponding to the 1.98-kilobase
XhoI-EcoO109IA fragment of NTP1; see Fig. 1)
labeled by random priming with [
32P]dCTP (Amersham
Pharmacia Biotech). Hybridizations were done at 65 °C in a solution
containing 5× saline/sodium phosphate/EDTA, 5× Denhardt and 0.5%
SDS, and 100 µg/ml tRNA. The filters were washed at 65 °C with
several changes of 1× saline, sodium phosphate, EDTA, 0.1% SDS
followed by 0.1× saline, sodium phosphate, EDTA, 0.1% SDS and exposed
for autoradiography. Between 106 and
107parasites were separated on SDS-polyacrylamide
electrophoresis gels, and the material was transferred onto
polyvinylidene difluoride membranes (Millipore). The filters were
probed with anti-HXGPRT antiserum (generously given by Buddy Ullman,
Portland, Oregon) or with anti-NTPase polyclonal antibody, GRA3, and
SAG1 monoclonal antibodies as described previously (2, 33). The primary
antibodies were generally used at 1:1000 dilution. Secondary anti-mouse
or anti-rabbit were horseradish peroxidase conjugates (Boehringer Mannheim) used at 1:2000 dilution followed by detection with ECL kit
(Amersham Pharmacia Biotech).
 |
RESULTS AND DISCUSSION |
We targeted the gene encoding the abundant NTPase in T. gondii for inhibition by antisense RNA. Previous experiments by
others suggest that a high ratio of transfected antisense RNA to the endogenous mRNA is required for effective reduction of expression of the target gene (34, 35). To increase the effectiveness of the
antisense expressing vector (Fig. 1), we
designed a construct that incorporated potentially enhancing features:
a potent NTP promoter (36), the inclusion of an autocatalytic ribozyme
structure and RNA structural elements that may act to stabilize the
antisense RNA, as well as the possible enrichment for antisense RNA
within the nuclear compartment (21, 37). Substituting the
polyadenylation signal with a cis-acting ribozyme that
cleaves in a site-specified manner leads to nuclear retention of the
product RNAs by generating export-deficient transcripts (21, 37). We
inserted a hammerhead ribozyme (23) immediately downstream of the
antisense DNA, thus replacing the 3'-untranslated region of the gene.
This operation potentially creates a pool of antisense RNA in the
nucleus comparable with the endogenous mRNA. Since 3'-end formation
promotes the export of mature polyadenylated RNA from the nuclear
compartment where antisense effects most likely occur in eukaryotes, we
did not include in these experiments a plasmid containing NTP antisense sequences with a polyadenylation signal (21, 22, 34, 35, 37).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 1.
Strategy for design of antisense-ribozyme
expression constructs. A, step 1. A 1.7-kilobase
fragment of the NTP 5'-flanking region was isolated from pBSNTP1 with
SpeI and BglII digests and subcloned at
SpeI-BamHI sites in the polylinker of pBS-RZ
(kindly provided by G. Carmichael). The resultant construct has the
entire 3'-untranslated region replaced by a hammerhead ribozyme and a
histone stem-loop structure, which has been included as a possible
stabilizing element against 3'-to-5' exonuclease degradation of
ribozyme-cleaved RNAs (23, 37). The histone-ribozyme sequence is
presented as a folded structure with the site of self-cleavage
indicated by a small arrow. A, step 2. A
1.98-kilobase XhoI-EcoO109IA fragment was excised
from the pBSNTP1, and the ends were filled in with the Klenow DNA
polymerase. The fragment was cloned in the antisense orientation into
the ribozyme cassette cleaved with EcoRI and filled in.
B, a 690-base pair polymerase chain reaction fragment
encompassing the coding region of HXGPRT derived from pminCAT vector
was blunt-ended and cloned into p5'NTPRZ in the antisense orientation
as described in A. Orientation of the coding region is
indicated by the arrowheads. Figs. are not drawn to scale.
Sp, SpeI; Xh, XhoI;
Bg, BglII, Av, AvrII,
RI, EcoRI, and EcoO,
EcoO109IA. Av and Bg were generated by
site-directed mutagenesis.
|
|
Clones expressing antisense NTP RNA (and hereafter referred to as
AS-NTP) displayed a dramatic reduction in NTPase enzymatic activity, ranging from ~4 to >19-fold (Fig.
2A). The variation in the
degree of inhibition as revealed by the enzymatic assay is considered a
general feature of antisense inhibition (20, 38). This variation may be
related to the difference in the steady-state levels of antisense RNA
generated due to chromosomal location of the gene or in copy number of
the integrated gene (38, 39). To ascertain that reduction in enzyme
activity was due to reduced synthesis of the protein, the amount of
NTPase protein was assessed by immunoblot. NTPase levels were
reduced between 86 to 88% when compared with WT in selected clones
expressing antisense RNA and correlated with the decrease in enzymatic
activity (Fig. 2B). To control for the possible nonspecific
effects of antisense RNA, we measured the levels of another dense
granule protein, GRA3, and of the surface antigen, SAG1. The level of each of these proteins was comparable in all the samples analyzed. By
immunofluorescence, NTPase signal was reduced in the transformants, whereas SAG1 expression was not affected (data not shown). The controls
in these experiments indicated that the expression of antisense NTP RNA
did not globally impair parasite metabolism.

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 2.
Antisense NTP RNA inhibits NTPase
expression. A, equal numbers of parasites were assayed
for NTPase activity as described under "Materials and Methods."
Values are expressed as % ATP hydrolyzed ±S.E. relative to control
(WT), which is taken as 100% (n = 6-10).
Values between the control (WT) and antisense-expressing
clones (AS-NTP) are significantly different
(p < 0.005) as determined by the two-tailed
t test. B, analysis of the steady-state levels of
protein of AS-NTP clones was done by immunoblot with equal numbers of
parasites using a polyclonal anti-NTPase antibody. Quantitation was
performed by densitometric scanning of autoradiographs, and values
obtained were expressed relative to WT, which is arbitrarily set as 1. AS-NTP 17, 12, 13, and 14 had values of 0.16, 0.12, 0.12, and 0.16 respectively. GRA3 and SAG1 were used as internal controls.
C, Northern blot analysis of steady-state levels of RNA was
performed with equal amounts of total RNA (10 µg/lane). As
judged by staining intensity on agarose gel (indicated by 18 S and 28 S
rRNA), the WT lane is underloaded; nevertheless, the band
corresponding to NTP is more intense than in lanes
containing the antisense clones. Values for clones AS-NTP 12, 17, and
14 as assessed had values of 0.16, 0.41, and 0.29, respectively,
relative to WT, which is set at 1. Note that NTP1 and
NTP3 genes (97% sequence similarity) encode two isoforms,
indistinguishable from each other by immunoblot (molecular mass = 63 kDa) and by Northern blot analysis (2.7 kilobases).
|
|
We determined the steady-state levels of NTP transcripts by Northern
blot analysis (Fig. 2C). NTP RNA levels were depressed at
least by 2- to 6-fold in clones expressing antisense RNA. There appeared to be a good correlation between the steady-state levels of
NTP RNA and the synthesis of the protein as assessed by Northern blot
and immunoblot, respectively. Using a double-stranded DNA probe in
Northern hybridization, we did not detect antisense RNA, although dot
blot analysis revealed the presence of the gene in the genomic DNA of
stably transfected clones (data not shown). One possible reason for
this is that the antisense transcripts or their cleavage products are
highly unstable and are degraded extremely rapidly in the parasite.
However, the reduced steady-state levels of NTP mRNA coupled with
the lack of detection of antisense RNA in the transfected clones is
consistent with observations in other systems (38-40). This is
generally assumed to be caused by duplex formation between antisense
RNA and the target mRNA in the nucleus, thereby interfering with
normal nuclear processing, cytoplasmic export, and translation of
mRNA (34, 35). A direct inhibition of the production of NTPase
through disruption of mRNA processing and transport seems plausible
since ribozyme-processed RNA has been shown to be retained in the
nuclear compartment (21, 37). These results indicate that antisense RNA
can block mRNA and protein expression in T. gondii.
Parasites expressing antisense RNA were impaired in intracellular
replication but not invasion. At 8 h after infection, equal numbers of parasitophorous vacuoles were established by both control (108 ± 4.9) and antisense-expressing clones (114 ± 7.2, 111 ± 14.3; Table I), indicating
that transgenic parasites that had a normal morphology invaded host
cells normally. However, once inside the cell, the transgenic parasites
divided more slowly than wild type, as evidenced by two different
assays for parasite replication: intracellular doubling (Table I; Fig.
3, A, B, and C) and nucleobase uptake (Fig. 3D). For example,
at 24 h after inoculation, the average doubling for the control
was 2.89, which differed from 2.17 and 1.67 (p < 0.01), displayed by the antisense-expressing parasites (Table I). For
the control parasites, this doubling corresponds to a replication time
of about 8 h, similar to that previously reported (30). The
reduced replication of the antisense parasites is coincident with
reduced [3H]uracil incorporated into parasite nucleic
acid pools as shown by the lower values (37 and 48%) in transformants
relative to wild type (Fig. 3D). The lag in replication of
the transformants in the first 24-32 h after infection may reflect the
deficit in initial NTPase levels and represents the time needed to
overcome this deficit by synthesis, packaging into dense granules, and exocytosis of a threshold amount of NTPase into the vacuolar space.
View this table:
[in this window]
[in a new window]
|
Table I
Distribution of parasitophorous vacuole sizes in antisense-expressing
and control parasites
Stably transfected transgenic clones and control RH parasites were
inoculated into fresh HFF cells. After 4 h, parasites that did not
infect were washed off, and the number of parasites per vacuole was
scored at the indicated times. The average number of vacuoles is
expressed as % and plotted in Fig. 3. Parasite doublings were
determined by log2(parasite number).
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 3.
Inhibition of parasite proliferation by
antisense RNA. The % distribution of vacuole sizes (number of
parasites/vacuole) was determined at 24 h (A) and
32 h (B) after infection with control and antisense
clones (also see Table I). Since all parasites within a single vacuole
replicate synchronously, the number of parasite doublings since
infection can be determined by log2(parasite number). At
time 24 h after infection, the median vacuole in the control
contained 8 parasites (corresponding to 3 parasite doublings), whereas
in the antisense clones, this was between 2 and 4 parasites (1-2
doublings), reflecting a delay in replication. Similarly, at 32 h,
the control had a median vacuole containing >8 parasites, whereas the
transgenic parasites still had 2-4 parasites. Data represent the
averages for at least 50 randomly selected vacuoles in four different
experiments. , control or WT; , AS-NTP 12; , AS-NTP 14. C, after infection, intracellular parasites were harvested
at the time points indicated and counted. AS-NTP 6, 12, 14, 18 over a
24-h period post-infection reproducibly replicated more slowly than the
control (p < 0.005). There were approximately half as
many parasites with AS-NTP clones as in the WT control at 24 h.
The doubling time as calculated from the data for the control was
8.2 h, whereas the AS-NTP clones had doubling times of 13, 14.5, 16, and 18 h, respectively. , control; , AS-NTP 17; ,
AS-NT 12; , AS-NTP 6; , AS-NTP 14. D, uracil
incorporation was assayed using HFF cells infected for 24 h as
under "Materials and Methods." Data are expressed relative to
control (which is taken as 100%) ±S.E. from three independent
experiments done in duplicate. Differences between values of the
control and experimental groups were statistically significant
(p < 0.005). Note that inhibition of parasite
replication is not a consequence of drug selection, because
pyrimethamine does not interfere with parasite replication (also see
RZ; Fig. 4D and Ref. 29).
|
|
To further control for nonspecific effects of antisense RNA, we
employed two additional constructs; the histone-ribozyme vector, p5'NTPRZ (Fig. 1A) and pAS-HXRZ, containing the HXGPRT
gene in the antisense orientation (Fig. 1B). Stable,
pyrimethamine-resistant lines expressing these constructs showed no
significant reduction in NTPase levels (Fig.
4A) nor in NTPase enzymatic
activity (clones AS-HXRZ-26, 32, and
RZ; Fig. 4C). In contrast, parasites harboring pAS-HXRZ displayed a profound reduction in HXGPRT levels (Fig. 4A), which was accompanied by a significant reduction in
HXGPRT activity (Fig. 4B). These transgenic parasites did
not reveal any morphological changes nor any growth retardation (Fig.
4D) and were comparable with the control HXGPRT knock out
strain of RH (KO; Fig. 4, C and D).
Similarly, parasite replication was not significantly reduced in
parasites harboring the p5'NTPRZ construct (RZ; Fig.
4D). The reduction in HXGPRT activity with no adverse
effects on parasite replication is consistent with the finding that
HXGPRT is not an essential gene (32). These results also
suggest that promoter interference or squelching is not responsible for
the inhibition of endogenous NTP expression. Although we have not
directly tested these constructs (Fig. 1) in host cells, it is unlikely
that they affect host cell growth or viability because they are driven
by the developmentally regulated NTP promoter (32). Therefore, we
conclude that the inhibition of parasite replication is a consequence
of a deficit in NTPase production brought about by antisense NTP
RNA.

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 4.
Inhibition of HXGPRT expression by antisense
RNA does not affect NTPase activity and parasite replication.
A, equal numbers of parasites were assayed by immunoblot
with a polyclonal antibody that recognizes bands corresponding to two
isoforms of HXGPRT (HXGPRT I = 26 kDa and HXGPRT II = 31 kDa)
as in Donald and Roos (29). Note that NTPase levels are comparable in
all samples. B, HXGPRT activity was expressed by % [3H]xanthine incorporation ±S.E. in two independent
experiments done in triplicate; activity from WT was taken as 100%.
C, NTPase activity was expressed as % ATP hydrolysis ±S.E.
from three independent experiments; activity from WT was taken as
100%. D, rate of [3H]uracil incorporation was
expressed relative to control (which is taken as 100%) ±S.E.
(n = 3-6). RZ, parasites harboring
p5'NTPRZ; KO, HXGPRT knock out strain (kindly provided by D. Roos (29)).
|
|
We propose that NTPase is functionally required for proliferation of
T. gondii. The molecular mechanism by which the reduction in
NTPase levels leads to inhibition of parasite replication is unknown.
Nevertheless, this study implicates NTPase for the first time in an
essential parasite function. Data from other studies support the notion
that NTPase may have a regulatory function in T. gondii
growth and replication. First, NTPase activity is tightly regulated in
vivo, as unfettered activation of the enzyme leads to rapid
exit of parasites from the host cell (8). Second, it has been shown
that quercetin (P-type ATPase inhibitor) effectively prevents growth of
T. gondii in culture at concentrations that inhibit NTPase
by >90% in vitro (41). Third, the secretion of NTPase into
the parasitophorous vacuoles occurs shortly before replication of
Toxoplasma sporozoites (infective forms within oocysts
generated after the sexual cycle in the definitive feline host) (42).
Finally, NTP promoter activity is developmentally down-regulated in
bradyzoites, the slowly dividing form of T. gondii that
persists in the host as a chronic infection (36). Of interest, in gene
knock out experiments we have never recovered any clones with a
disrupted NTP1 or NTP3 gene locus, although we
have employed targeting vectors with large contiguous genomic sequences. This suggests indirectly that NTPase is an essential gene
and may thus provide a potential target for chemotherapy in T. gondii.
The experiments were undertaken in this study to demonstrate the
biological role of NTPase, one of the most potent ATP degrading enzymes, in the metabolism of the parasite. To this end, we employed antisense RNA coupled to a hammerhead ribozyme to reveal the role of
NTPase in parasite proliferation. Although it remains to be formally
proven, a complete abrogation of NTPase expression may be incompatible
with parasite growth and survival. Since NTPase can also influence
parasite exit from the host cell (8), these findings suggest that the
physiological role of NTPase may be multifunctional. In this study, we
have also shown that even a very abundant protein such as NTPase or
HXGPRT is titratable by antisense RNA. Because it is effective and
simple, antisense RNA strategy should find widespread application among
apicomplexan parasites for the rapid analysis of gene function. For
example, genes that are refractory to targeted gene disruption or
essential genes that generate a lethal phenotype when they are
disrupted, are especially amenable to this strategy.
 |
ACKNOWLEDGEMENTS |
We thank G. Carmichael, Storrs, CT for
generously donating pBS-RZ and Buddy Ullman, Oregon Health Sciences
University for HXGPRT antibody. We also thank Heinreich Hoppe,
Huân Ngô, Christian Tschudi, and Elisabetta Ullu for
critically reading the manuscript.
 |
FOOTNOTES |
*
This work was supported by a National Institutes of Health
Public Health Service Grant from the (to K. A. J.), a National Institutes of Health Minority Supplement Award (to V. N.), and the
Markey Physician Scientist Training program (to B. U. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence and reprint requests should be addressed:
Infectious Diseases Section, Dept. of Internal Medicine, Yale
University School of Medicine, 808 LCI, 333 Cedar St., New Haven, CT
06520-8022. Tel.: 203-785-4140; Fax: 203-785-3864; E-mail: keith.joiner{at}yale.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
NTPase, nucleoside
triphosphate hydrolase;
HXGPRT, hypoxanthine-xanthine-guanine
phosphoribosyl transferase;
GRA3, dense granule antigen 3;
SAG1, surface antigen 1;
HFF, human foreskin fibroblasts;
AS-, antisense;
WT, wild type.
 |
REFERENCES |
-
Luft, B. J.,
and Remington, J. S.
(1992)
Clin. Infect. Dis.
15,
211-222[Medline]
[Order article via Infotrieve]
-
Bermudes, D.,
Peck, K. R.,
Afifi-Afifi, M.,
Beckers, C. J. M.,
and Joiner, K. A.
(1994)
J. Biol. Chem.
269,
29252-29260[Abstract/Free Full Text]
-
Asai, T.,
Miura, S.,
Sibley, D. L.,
Okabayashi, H.,
and Tsutomu, T
(1995)
J. Biol. Chem.
270,
11391-11397[Abstract/Free Full Text]
-
Asai, T.,
O'Sullivan, W. J.,
and Tatibana, M.
(1983)
J. Biol. Chem.
258,
6816-6822[Abstract/Free Full Text]
-
Carruthers, V.,
and Sibley, D.
(1997)
Eur. J. Cell Biol.
73,
114-123[Medline]
[Order article via Infotrieve]
-
Schwab, J. C.,
Beckers, C. J. M.,
and Joiner, K. A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
509-513[Abstract]
-
Sibley, L. D.,
Niesman, I. R.,
Asai, T.,
and Takeuchi, T.
(1994)
Exp. Parasitol.
79,
301-311[CrossRef][Medline]
[Order article via Infotrieve]
-
Silverman, J. A.,
Qi, H.,
Riehl, A.,
Beckers, C.,
Nakaar, V.,
and Joiner, K. A
(1998)
J. Biol. Chem.
273,
12352-12359[Abstract/Free Full Text]
-
Kim, K. D.,
Soldati, D.,
and Boothroyd, J. C.
(1993)
Science
262,
911-914[Medline]
[Order article via Infotrieve]
-
Donald, R. G. K.,
and Roos, D.
(1998)
Mol. Biochem. Parasitol.
91,
295-305[CrossRef][Medline]
[Order article via Infotrieve]
-
Menard, R.,
Sultan, A. A.,
Cortes, C.,
Altszuler, R.,
van Dijk, M. R.,
Janse, C. J.,
Waters, A. P.,
Nussenzweig, R. S.,
and Nussenzweig, V.
(1997)
Nature
385,
336-340[CrossRef][Medline]
[Order article via Infotrieve]
-
Green, P. J.,
Pines, O.,
and Inouye, M.
(1986)
Annu. Rev. Biochem.
55,
569-597[CrossRef][Medline]
[Order article via Infotrieve]
-
Knecht, D. A.,
and Loomis, W. F.
(1987)
Science
236,
1081-1086[Medline]
[Order article via Infotrieve]
-
Zhang, W-W.,
and Matlashewski, G.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
8807-8811[Abstract/Free Full Text]
-
Cabrera, C. V.,
Alonso, M. C.,
Johnston, P.,
Phillips, R. G.,
and Lawrence, P. A.
(1987)
Cell
50,
659-663[Medline]
[Order article via Infotrieve]
-
Melton, D. A.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
144-148[Abstract]
-
Bevilacqua, A,
Erickson, R. P.,
and Hieber, V.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
831-835[Abstract]
-
Ecker, J. R.,
and Davis, R. W.
(1986)
Proc. Natl. Acad. Sc. U. S. A.
83,
5372-5376[Abstract]
-
Rothstein, S. J.,
DeMaio, J.,
Strand, M.,
and Rice, D.
(1987)
Proc. Natl. Acad. Sci. U. S. A.
84,
8439-8443[Abstract]
-
van der Krol, A. R.,
Lenting, P. E.,
Veenstra, J.,
van der Meer, I. M.,
Koes, R. E.,
Gerats, A. G. M.,
Mol, J. N. M.,
and Stuitje, A. R.
(1988)
Nature
333,
866-869[CrossRef]
-
Eckner, R.,
Ellmeier, W.,
and Birnstiel, M.
(1991)
EMBO J.
10,
3513-3522[Abstract]
-
Cornelissen, M.
(1989)
Nucleic Acids Res.
17,
7203-7209[Abstract]
-
Haseloff, J.,
and Gerlach, W. L.
(1988)
Nature
334,
585-591[CrossRef][Medline]
[Order article via Infotrieve]
-
Gu, J-L.,
Veerapanane, D.,
Rossi, J.,
Natarajan, R.,
Thomas, L.,
and Nadler, J.
(1995)
Circ. Res.
77,
14-20[Abstract/Free Full Text]
-
Taylor, N. R.,
Kaplan, B. E.,
Swidersk, P.,
Lin, H.,
and Rossi, J. J.
(1992)
Nucleic Acids Res.
20,
4559-4564[Abstract]
-
Montgomery, R. L.,
and Dietz, H. C.
(1997)
Hum. Mol. Genet.
6,
519-525[Abstract/Free Full Text]
-
Cech, T. R.
(1988)
J. Am. Med. Assoc.
260,
3030-3034[Abstract]
-
Sarver, M.,
Cantin, E.,
Chang, P.,
Ladne, P.,
Stephens, D.,
Zaia, J.,
and Rossi, J.
(1990)
Science
247,
1222-1225[Medline]
[Order article via Infotrieve]
-
Donald, R. G. K.,
and Roos, D. S.
(1993)
Proc. Natl. Acad. Sci.
90,
11703-11707[Abstract]
-
Fichera, M. E.,
Bhopale, M. K.,
and Roos, D. S.
(1995)
Antimicrob. Agent Chemother.
39,
1530-1537[Abstract]
-
Pfefferkorn, E. R.,
Nothnagel, R. F.,
and Borotz, S. E.
(1992)
Antimicrob. Agents Chemother.
36,
1091-1096[Abstract]
-
Donald, R. G. K.,
Carter, D.,
Ullman, B.,
and Roos, D. S.
(1996)
J. Biol. Chem.
271,
14010-14019[Abstract/Free Full Text]
-
Ossorio, P. N.,
Dubremetz, J.-F.,
and Joiner, K. A.
(1994)
J. Biol. Chem.
269,
15350-15357[Abstract/Free Full Text]
-
Kim, S. K.,
and Wold, B. J.
(1985)
Cell
42,
129-138[Medline]
[Order article via Infotrieve]
-
Izant, J. G.,
and Weintraub, H.
(1985)
Science
229,
345-352[Medline]
[Order article via Infotrieve]
-
Nakaar, V.,
Bermudes, D.,
Peck, K. R.,
and Joiner, K. A.
(1998)
Mol. Biochem. Parasitol.
92,
229-239[CrossRef][Medline]
[Order article via Infotrieve]
-
Liu, Z.,
Batt, D. B.,
and Carmichael, G. G.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
4258-4262[Abstract]
-
Smith, C. J. S.,
Watson, C. F.,
Ray, J.,
Bird, C. R.,
Morris, P. C.,
Schuch, W.,
and Grierson, D.
(1988)
Nature
334,
724-726[CrossRef]
-
Rodermel, S. R.,
Abbott, M. S.,
and Bogorad, L.
(1988)
Cell
55,
673-681[Medline]
[Order article via Infotrieve]
-
Kuipers, A. G. J.,
Soppe, W. J. J.,
Jacobsen, E.,
and Visser, R. G. F.
(1995)
Mol. Gen. Genet.
246,
745-755[Medline]
[Order article via Infotrieve]
-
Asai, T. & Sibley, L. D. (1996) Fourth International
Biennial Toxoplasma Conference, July 22-26, 1996 (abstract),
Drymen, Scotland
-
Tilley, M.,
Fichera, M. E.,
Jerome, M. E.,
Roos, D. S.,
and White, M. W.
(1997)
Infect. Immun.
65,
4598-4605[Abstract]
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.