©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Rat Antizyme Inhibits the Activity but Does Not Promote the Degradation of Mouse Ornithine Decarboxylase in Trypanosoma brucei(*)

Shao-bing Hua (1), Xianqiang Li (2), Philip Coffino (2) (3), Ching C. Wang (1)(§)

From the (1) Departments of Pharmaceutical Chemistry, (2) Microbiology and Immunology, and (3) Medicine, University of California, San Francisco, California 94143

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Ornithine decarboxylase (ODC) of African trypanosomes is an important target for anti-trypanosomal chemotherapy because of its remarkable stability in vivo. The in vivo activity and stability of mammalian ODC are regulated by polyamines. Polyamines induce antizyme, which inactivates ODC by tight association and promotes degradation of ODC by the mammalian 26 S proteasome. Here we found, in contrast to mammalian cells, that polyamines caused no reduction of ODC activity in Trypanosoma brucei. Mouse ODC expressed in T. brucei was also unaffected by exogenous polyamines, suggesting that a mammalian antizyme equivalent may be absent in T. brucei. The rat antizyme expressed in T. brucei was found capable of inhibiting mouse ODC activity by the formation of rat antizyme-mouse ODC complex. However, complex formation did not lead to degradation of mouse ODC in T. brucei. Further in vitro experiments suggested the presence of an inhibitory factor(s) in trypanosome, which interferes with the degradation of mouse ODC. We also demonstrated the presence of proteasomes in T. brucei. But the mobility of the trypanosomal proteasome on native gel is different from that of the mammalian proteasome. Thus, the absence of antizyme, the presence of inhibitory factor(s), and the differences between trypanosomal and mammalian proteasome may account for the stability of mouse ODC in T. brucei cells.


INTRODUCTION

Ornithine decarboxylase (ODC() ; L-ornithine carboxylase, EC 4.1.1.17) is the key enzyme in de novo synthesis of polyamines, which are ubiquitous in and essential for cells (1, 2) . The inhibition of ODC has been investigated as a potential way to control rapid cell proliferation and to cure certain infectious deceases (3) . Indeed, -DL-difluoromethylornithine (DFMO), a catalytic, irreversible inhibitor of ODC, has been found to cure infections of Trypanosoma brucei in mice and Trypanosoma gambiense in man (4, 5) . DFMO depletes the pool of putrescine and spermidine in African trypanosomes, resulting in cessation of cell growth and eventual elimination of the parasites by the host immune response (5, 6) . The anti-trypanosomal action of DFMO can be reversed by intraperitoneal injections of polyamines in the murine model (7) . The selective DFMO toxicity toward the trypanosomes may be attributable to a difference in the in vivo stabilities between trypanosomal ODC and its mammalian counterpart (8) . Mammalian ODC is one of the most short lived cellular proteins in vivo with a half-life of 10-50 min (1, 9) . On the other hand, we found that the trypanosomal ODC is very stable in T. brucei, presumably because a PEST sequence present at the carboxyl terminus of mouse ODC is absent from T. brucei ODC (10) . This carboxyl-terminal PEST sequence could be implicated in the rapid intracellular turnover of mammalian ODC. Truncation of the 37 residues at the carboxyl terminus of mouse ODC stabilized the protein in vivo (11) , and addition of the carboxyl terminus of mouse ODC to trypanosomal ODC led to the rapid degradation of the chimeric protein in Chinese hamster ovary cells (12) , indicating that the sequence at the mouse ODC carboxyl terminus plays an important role in bringing about its rapid degradation in vivo. However, when full-length mouse ODC was expressed in the procyclic form of T. brucei, we found that the mouse enzyme was stable (13) . This observation suggests the absence and/or the inhibition of necessary machineries in T. brucei cells required for rapid degradation of mammalian ODC. We decided therefore to look into the mechanisms behind the unusual stability of mouse ODC in the procyclic form of T. brucei.

In mammalian cells, a noncompetitive inhibitory protein named antizyme is involved in the degradation of ODC both in vivo and in vitro (14, 15, 16) . The expression of antizyme in mammalian cells can be induced by polyamines (9) . Antizyme inhibits ODC activity by forming an ODC-antizyme complex (9, 14, 17) . The complex thus formed causes the exposure of the carboxyl terminus of ODC (18) , which accelerates the degradation of ODC by the 26 S proteasome in an ATP-dependent but ubiquitin-independent manner (19, 20, 21) . The 26 S proteasome is a large complex consisting of two functionally distinct units: a central cylindrical 20 S proteasome with at least three distinct protease activities, namely trypsin-like, chymotrypsin-like, and peptidylglutamyl-peptide hydrolase activities, respectively; and regulatory parts that attach to the central core (22, 23, 24) . The mammalian 20 S proteasome contains 20-25 different proteins with molecular sizes of 22-34 kDa resolved on a two-dimensional gel (24) . The manifestation of multiple catalytic activities at independent active sites within a single enzyme complex may be advantageous for the rapid and selective degradation and inactivation of various types of cellular proteins. In addition to the selective ubiquitin-independent degradation of ODC, the 26 S proteasome performs selective degradation of regulatory proteins related to cell cycle progression through a pathway that requires substrate ubiquitination (25, 26) . The 26 S proteasome is also thought to play other roles in intracellular proteolysis such as antigen presentation (23) .

In this study we first examined whether an antizyme is present in T. brucei and found it to be absent. We then expressed both mouse ODC and rat antizyme in T. brucei by DNA transformation and noted the anticipated rat antizyme-mediated inactivation of mouse ODC enzymatic activity through antizyme-ODC complex formation. However, formation of the complex does not lead to rapid degradation in T. brucei, which could be attributed to the presence in T. brucei of an inhibitor(s) of proteasome function. We have also, for the first time, identified the proteasomes in T. brucei, which turn out to be distinct from mammalian proteasomes.


EXPERIMENTAL PROCEDURES

Cell Cultures and Ornithine Decarboxylase Activity Assay

Procyclic cells of a cloned T. brucei brucei strain TREU667 were continuously cultivated at 26 °C by serial passages in Cunningham's medium (27) supplemented with 10% fetal bovine serum (Gemini Bioproducts, Inc.). The hepatoma cells (HTC) were cultured as described previously (14) . The trypanosomal cell suspension was diluted with fresh Cunningham's medium containing fetal bovine serum to a density of approximately 1 10cells/ml and incubated at 26 °C overnight before the ODC activity assay. Cells were washed with cold phosphate-buffered saline, lysed, and assayed for ODC activity essentially as described previously (14) . One unit of ODC activity is defined as 1 nmol of COreleased from L-ornithine/h. One unit of antizyme activity is defined as the amount of antizyme required to inhibit 1 unit of ODC activity.

Plasmid Construction and Transformation of T. brucei

The rat antizyme partial cDNA clone, Z1 (28, 29) , was obtained from Dr. S. Hayashi. Due to the absence of a suitably placed methionine initiation codon in the clone, an in-frame ATG codon was added at the immediate 5`-end of the clone. The antizyme thus expressed from this construct in Chinese hamster ovary cells is known to execute all of the expected cellular and biochemical functions (30) . An XhoI site was generated upstream of the ATG codon in the primer sequence (5`-AAAACTCGAGCATATGCCAGAGAGAAAGAAGGGGA) for polymerase chain reaction. At the 3`-end of the clone, a BamHI site was added upstream of the poly(A) tail (primer sequence: 5`-TTTTGGATCCAGATCACTTTATTTATTAG). The XhoI/ BamHI fragment of the polymerase chain reaction product was inserted into the XhoI/ BamHI sites of the vector pTSA-Hyg2 (31) , which contains a hyg gene whose expression in T. brucei is under the control of a procyclic acidic repetitive protein promoter. The nucleotide sequence of the insert was confirmed by sequencing. The plasmid thus constructed was named pAZ. Plasmids pMOD300 and pTOD300 containing the inserts of full-length cDNA encoding mouse ODC and T. brucei ODC, respectively, were constructed previously from a similar vector pTSA-Neo2, which has a neo gene controlled by the procyclic acidic repetitive protein promoter (13) . The DNAs of pAZ, pMOD300, and pTOD300 were each linearized at the MluI site at the center of T. brucei tubulin intergenic region and electroporated into the T. brucei cells (31) either individually or in combination. The stable transformants were selected in the Cunningham's medium containing 10% fetal bovine serum in the presence of G418 (geneticin, Life Technologies, Inc.) (50 µg/ml), or hygromycin (Calbiochem-Novabiochem Corporation) (50 µg/ml), or both.

Southern, Northern, and Slot Blot Hybridizations

Cellular DNA was isolated from the procyclic forms of T. brucei according to Hua and Wang (32) . Total cellular RNA was isolated as described (33) , and the poly(A) RNA was prepared by using the Fast-track mRNA isolation system (Invitrogen). The Southern, Northern, and slot blot hybridizations were carried out as described previously (32) .

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunostaining

Cells were lysed in the lysis solution (2% (w/v) SDS; 125 mM Tris-HCl, pH 6.8; 5% (v/v) -mercaptoethanol; 20% (v/v) glycerol). The lysate was centrifuged at 16,000 g at 4 °C for 10 min. The supernatant was fractionated on a 12.5% SDS-PAGE. The separated proteins were electroblotted onto polyvinylidene difluoride Immobilon membrane (Millipore Inc.), immunostained, and visualized with the enhanced chemiluminescence system (Amersham Corp.) as described previously (32) .

In Vivo Pulse-Chase Labeling of Protein and Immunoprecipitation

The trypanosome cells were washed twice with cysteine/cystine-free Cunningham's medium, then suspended in the same medium containing L-[S]cysteine (250 µCi/ml, 600 Ci/mmol) (DuPont NEN) to a density of 5-6 10cells/ml. After incubation at 26 °C for 1 h, the radioactive medium was replaced with the regular Cunningham's medium and incubation continued. Cells were collected at time points of 0, 2, 4 and 6 h. Alternatively, cells were collected immediately after the L-[S]cysteine labeling without chasing. The cells were lysed in RIPA buffer (150 mM NaCl; 0.1% SDS; 0.5% sodium deoxycholate; 1% (v/v) Nonidet P-40; 50 mM Tris-HCl, pH 8.0) (34) , and pretreated with a rabbit preimmune serum. Mouse ODC, trypanosomal ODC, and rat antizyme were immunoprecipitated with their respective antisera (see below) and Pansorbin (formalin-fixed Staphylococcus aureus cells) (Calbiochem-Novabiochem) following a standard protocol (34) . HTC cells were labeled in culture medium containing L-[S]cysteine as described previously (30) , and ODC was immunoprecipitated as above. The immunoprecipitated proteins were subjected to 12.5% SDS-PAGE and autoradiography.

In Vitro ODC Inhibition Assay

Antizyme was produced by the in vitro transcription/translation systems described by Li and Coffino (18) . Five microliters of the antizyme thus generated (containing approximately 0.25 unit of antizyme) was mixed with 0.5 unit of purified native mouse ODC and incubated on ice for 10 min. Alternatively, lysates from 1.5 10pAZ-transformed T. brucei cells were mixed with 0.5 unit of purified native mouse ODC and kept on ice for 10 min. These mixtures were then assayed for ODC activity as described above.

In Vitro ODC Degradation Assay

Mouse ODC and antizyme proteins were prepared by the in vitro transcription/translation system as described previously (30) . Mouse ODC was labeled with L-[S]methionine during the in vitro translation and mixed with in vitro translated but unlabeled antizyme followed by the addition of an ATP-regenerating system as described previously by Li and Coffino (18) . Alternatively, the in vitro translated, unlabeled antizyme sample was replaced with various trypanosomal cell lysates (1.5 10cells) for the degradation assay in an attempt to search for activity capable of degrading mouse ODC in the lysates. Treated mouse ODC was separated by SDS-PAGE and visualized by autoradiography.

Purification and Analysis of Proteasomes

Proteasomes were isolated from trypanosome and rabbit blood cells according to Hoffman et al. (35) with some modifications. Briefly, cells were lysed in 10 mM Tris-HCl, pH 7, 1 mM dithiothreitol, 1 mM ATP, 0.1 mM EDTA by sonication in an ice-ethanol bath. Cell breakage was monitored under a microscope. After centrifuging the lysate at 80,000 g for 60 min, glycerol was added to the supernatant to 20% (v/v). The lysate was then loaded onto a DEAE-cellulose (Sigma) column equilibrated in the TSDG buffer (10 mM Tris-HCl, pH 7; 25 mM KCl; 10 mM NaCl; 1.1 mM MgCl; 0.1 mM EDTA; 1 mM dithiothreitol; 20% glycerol). The column was washed with 5 bed volumes of TSDG and 5 bed volumes of TSDG containing 100 mM KCl and eluted finally with TSDG containing 250 mM KCl. The eluate was then centrifuged at 100,000 g for 15 h at 4 °C. The pellet was resuspended in TSDG and subjected to electrophoresis in a nondenaturing gel for 500 V-h as described (35) . The protease activity in the gel was detected with a mixture of two fluorogenic peptides in 30 mM Tris-HCl, pH 7.8; 5 mM MgCl; 10 mM KCl; 0.5 mM dithiothreitol; 2 mM ATP for 30 min at 37 °C. The fluorogenic peptides used were 20 µM Suc-Leu-Leu-Val-Tyr-MCA (4-methyl-coumaryl-7-amide) and 200 µM Pro-Phe-Arg-MCA (Sigma). The fluorescent gel was photographed and then stained in 0.2% Coomassie Brilliant Blue. Alternatively, the fluorescent bands were excised and soaked in 125 mM Tris-HCl, pH 6.8; 2% (w/v) SDS; 5% (v/v) -mercaptoethanol; 20% (v/v) glycerol for 15 h at 37 °C. The samples were then subjected to SDS-PAGE and immunostaining with rabbit anti-human 20 S proteasome antibodies as described above (35) .

Other Methods

Autoradiographic and immunostaining results were analyzed with a laser densitometer (LKB model 2202 Ultrascan). Protein concentrations were determined with protein assay reagent from Bio-Rad using bovine immunoglobulin (IgG) as a standard.

Materials

Putrescine and all of the chemicals for making Cunningham's medium were tissue culture reagents from Sigma. Spermidine was from Calbiochem. The rabbit antiserum against antizyme was raised using the glutathione S-transferase-antizyme fusion protein (30) . Rabbit antiserum against trypanosomal ODC was prepared from purified recombinant T. brucei ODC (8) . Rabbit anti-mouse ODC antiserum was a gift from Dr. O. Jänne of the Rockefeller University. Rabbit antibody against human 20 S proteasome was a generous gift from Dr. M. Rechsteiner of the University of Utah (35) . Donkey anti-rabbit IgG-horseradish peroxidase conjugate was a product from Amersham.


RESULTS

Effect of Polyamines on T. brucei ODC Activity

Polyamines such as putrescine and spermidine regulate ODC activities in many living organisms including mammals (9) , Xenopus (36) , yeast (37) , and Neurospora (38) . The presence of excessive polyamines results in dramatic reductions of ODC activities. We thus investigated the potential effects of polyamines on the ODC activity of procyclic T. brucei grown in culture medium. As shown in Fig. 1A, supplementation of the growth medium with either putrescine or spermidine up to 5 mM resulted in no apparent decrease in ODC activity in T. brucei over a period of 6 h. This is in sharp contrast to the results with rat HTC cell cultures, in which the ODC activity was markedly reduced upon the addition of putrescine (Fig. 1 B). It has been shown that putrescine and spermidine can effectively enter T. brucei cells (39) . These results thus indicate that there is no induced inhibition of trypanosomal ODC activity by putrescine or spermidine, in contrast to all eukaryotic cells examined previously.


Figure 1: Effect of polyamines on the activity of ODC. Cells of panel A, T. brucei ( Tb), panel B, HTC cultures, and panel C, T. brucei transformed with mouse ODC cDNA ( Tb/MOD) were incubated in the absence or presence of either 5 mM putrescine ( putr.) or 5 mM spermidine ( sper.) for the indicated time and lysed for ODC activity assays.



Trypanosomal ODC Is Stable in Polyamine-treated T. brucei

In mammalian cells and Xenopus, the synthesis of an inhibitory protein antizyme can be induced by the presence of polyamines (9, 17, 36) . Antizyme reduces ODC activity dramatically and destabilizes ODC protein in mammalian cells (15, 16) . Antizyme was also shown to interact with mouse ODC protein in vitro, inhibit ODC activity, and accelerate its degradation (14, 18, 30) . In the above section, we observed no ODC activity change in the polyamine-treated T. brucei. Stable activity may result from the balance of both increased degradation and increased synthesis of trypanosomal ODC in the treated T. brucei. We therefore examined directly the stability of ODC protein in the treated trypanosomal cells. T. brucei cells were pulse labeled with L-[S]cysteine and then chased in medium supplemented with 5 mM putrescine. The radiolabeled trypanosomal ODC was immunoprecipitated and analyzed by SDS-PAGE and autoradiography. The 45-kDa T. brucei ODC remained stable during the 6-h chase period (Fig. 2 A). There was no obvious change in migration in SDS-PAGE of the ODC from T. brucei samples treated with putrescine. On the other hand, the radiolabeled mouse ODC protein was degraded very rapidly in HTC cells upon incubation in the medium containing putrescine (Fig. 2 B), as observed previously (16) . Slot blot and Northern hybridizations of T. brucei poly(A) RNA with a rat antizyme cDNA probe provided no indication of the presence of T. brucei transcripts homologous to the probe (see Fig. 4). These results suggest that antizyme is absent in T. brucei.


Figure 2: L-[S]Cysteine pulse-chase and immunoprecipitation. T. brucei cells ( panel A) or mouse HTC cells ( panel B) were pulse labeled with [S]cysteine as described under ``Experimental Procedures'' and chased for the indicated time period with nonradioactive cysteine and in the presence of 5 mM putrescine. The cells were lysed, and T. brucei ODC ( panel A) and mouse ODC ( panel B) were immunoprecipitated with their respective antisera. The proteins were then analyzed by SDS-PAGE and autoradiography. Lane m in panel B is the in vitro translated product of mouse ODC. The arrowhead in panel A indicates the band of T. brucei ODC; the arrow in panel B points the mouse ODC.




Figure 4: Northern analysis of rat antizyme expressed in T. brucei. Panel A, poly(A)RNA (0.5 µg) isolated from T. brucei stable transformants was separated on a denaturing agarose gel, transferred onto a nitrocellulose membrane, and hybridized with the rat antizyme cDNA probe. Lane 1, Tb/TSA-Hyg2; lane 2, Tb/AZ; lane 3, Tb/TOD-AZ; lane 4, Tb/MOD-AZ. The probe was washed off the blot, and the same blot was hybridized with T. brucei -tubulin probe afterward ( panel B).



Expression of Rat Antizyme in T. brucei

We have previously expressed mouse ODC in its native form in T. brucei by DNA transformation (13) (the transformant is herein designated Tb/MOD). Mouse ODC isolated from Tb/MOD possesses all of the in vitro biochemical properties identified in native mouse ODC, but it has little turnover in the trypanosomal cells (13) . In the present investigation, the Tb/MOD cells were incubated with polyamines (putrescine and spermidine) and assayed for ODC activity. Polyamine treatment caused no detectable change of the mouse ODC activity (Fig. 1 C), thus providing further evidence that T. brucei lacks an antizyme responsive to polyamines. To investigate whether this stability of mouse ODC in T. brucei cells was indeed solely due to the absence of antizyme in T. brucei, we transformed the T. brucei procyclic cells with rat antizyme cDNA in vector pTSA-Hyg2 (resulting in transformant Tb/AZ) and selected for stable transformants in the presence of hygromycin B. Similarly, each of the two stable transformants Tb/MOD and Tb/TOD which overexpress mouse ODC and T. brucei ODC, respectively, under the control of a different vector, pTSA-Neo2 (13) , was also transformed with rat antizyme cDNA in the pTSA-Hyg2 vector and selected under the double pressure of G418 and hygromycin B. The two stable double transformants were designated Tb/MOD-AZ and Tb/TOD-AZ, respectively. As controls, wild type T. brucei, Tb/MOD and Tb/TOD were each transformed with the pTSA-Hyg2 vector without any inserts, generating Tb/TSA, Tb/MOD-TSA, and Tb/TOD-TSA, respectively. All of the transformants were then assayed for ODC activity in the cell lysates. As shown in Fig. 3, the rat antizyme caused a reduction of total mouse ODC activity by 20-25% in Tb/MOD-AZ compared with the activities from Tb/MOD and Tb/MOD-TSA. However, the antizyme had no apparent effects on the trypanosomal ODC activities (compare Tb/TOD, Tb/TOD-TSA, and Tb/TOD-AZ in Fig. 3). Southern analyses (data not shown) indicated that the antizyme cDNA has been successfully integrated into the T. brucei genome in all of the stable transformants. Northern hybridizations (Fig. 4) demonstrated that in each case the integrated antizyme DNA was transcribed in the poly(A) RNA form at the expected size, presumably correctly processed. Furthermore, Western blot analysis confirmed that the antizyme protein was successfully expressed in the transformants and migrated at expected for a protein of its size, 26.5 kDa (Fig. 5). To quantitate the level of protein accumulation of mouse ODC expressed in Tb/MOD-AZ, we analyzed Tb/MOD-AZ cell extracts by immunoblot, using purified native mouse ODC as a standard. The signals on the immunoblots were quantitated by densitometric analysis. The accumulation level of rat antizyme expressed in Tb/MOD-AZ was similarly examined, using the purified Escherichia coli-expressed rat antizyme protein as a standard. The results (not shown) revealed that mouse ODC protein accumulates at approximately 1.7 pmol/10cells, whereas antizyme is present at approximately 1.5 pmol/10cells. Thus, rat antizyme was successfully expressed in T. brucei, and it partially reduced mouse ODC activity in the trypanosomal cells.


Figure 3: ODC activities of T. brucei and its stable transformants. Tb, T. brucei; TSA, Tb/TSA-Hyg2 (vector) transformant; AZ, Tb/AZ transformant (overexpressing rat antizyme); TOD, Tb/TOD transformant (overexpressing trypanosome ODC); TOD-TSA, Tb/TOD-TSA double transformant; TOD-AZ, Tb/TOD-AZ double transformant (overexpressing both trypanosome ODC and rat antizyme); MOD, Tb/MOD transformant (overexpressing mouse ODC); MOD-TSA, Tb/MOD-TSA double transformant; MOD-AZ, Tb/MOD-AZ double transformant (overexpressing both mouse ODC and rat antizyme).




Figure 5: Immunostaining of rat antizyme expressed in T. brucei. Panel A, cell lysates from 1 10cells each were separated by SDS-PAGE and immunostained with the rabbit antiserum against glutathione S-transferase-rat antizyme fusion protein. From left to right, first lane, T. brucei ( Tb); second lane, Tb/TSA-Hyg2 ( Tb/TSA); third lane, Tb/AZ; fourth lane, Tb/TOD; fifth lane, Tb/TOD-TSA; sixth lane, Tb/TOD-AZ; seventh lane, Tb/MOD; eighth lane, Tb/MOD-TSA; ninth lane, Tb/MOD-AZ. Panel B, as a control, cell lysates from 1 10cells each were immunostained with mouse monoclonal antibody against chicken -tubulin. The upper arrowhead in panel A indicates the nonspecific signals, and the lower arrowhead indicates the antizyme band.



Rat Antizyme Is Associated with Mouse ODC but Does Not Promote Its Degradation in T. brucei

To distinguish whether inhibition or degradation accounts for capacity of antizyme to reduce mouse ODC activity, we first determined whether the two proteins are associated in trypanosomal cells. We labeled cells expressing these proteins with [S]cysteine and immunoprecipitated with antibodies directed against antizyme or against ODC. As shown in Fig. 6A, antiserum against rat antizyme coimmunoprecipitated mouse ODC along with antizyme from the lysate of Tb/MOD-AZ. Similarly, antiserum against mouse ODC precipitated rat antizyme as well as mouse ODC. Densitometric analysis revealed that approximately 20-25% of the total mouse ODC was coprecipitated from the Tb/MOD-AZ lysate by the antiserum against rat antizyme, and about 20-25% of the total rat antizyme was coprecipitated by the antiserum against mouse ODC, in agreement with the previously observed extent of inhibition of mouse ODC activity in the same lysate (Fig. 3). Furthermore, densitometry also indicated that the autoradiographic signal of MOD is approximately 3-fold stronger than that of AZ. Because mouse ODC contains 12 cysteine residues (40) , whereas AZ contains only 4 cysteine residues (29) , this implies that both MOD and AZ are expressed at an equal molar ratio in Tb/MOD-AZ. However, the antiserum against trypanosomal ODC did not coprecipitate any detectable antizyme, nor did the antiserum against antizyme coprecipitate any trypanosomal ODC from the lysate of Tb/TOD-AZ (Fig. 6 A). These results suggest that rat antizyme could bind to mouse ODC but not to trypanosomal ODC in T. brucei cells, and this association probably led to the inhibition of mouse ODC activity.


Figure 6: Rat antizyme is associated with mouse ODC but does not promote its degradation in T. brucei. Panel A, T. brucei cells (2 10) were labeled with L-[S]cysteine, lysed, and immunoprecipitated with antisera. The immunoprecipitants were separated by SDS-PAGE and visualized by autoradiography. The cell lysates of different transformants were immunoprecipitated with either the antiserum against glutathione S-transferase-rat antizyme fusion protein ( -A) or the antiserum against mouse ODC ( -M), or the antiserum against T. brucei ODC ( -T). The arrowhead on the left indicates the nonspecific cross-reaction. Panel B, pulse-chase experiment. Tb/MOD-AZ cells were pulse labeled with [S]cysteine, chased with nonradioactive cysteine for the indicated time period, and lysed. The lysates were then immunoprecipitated with antiserum against mouse ODC and analyzed by SDS-PAGE and autoradiography. The arrowhead on the right indicates the mouse ODC band.



On the other hand, when the cells of the trypanosomal transformant containing both mouse ODC and antizyme ( Tb/MOD-AZ) were subjected to [S]cysteine pulse-chase experiment followed by immunoprecipitation with antiserum against mouse ODC, mouse ODC was seen to be very stable (Fig. 6 B). No sign of degradation of mouse ODC was observed during an extended 6-h chase period. These results indicate that although the rat antizyme is associated with mouse ODC and causes the reduction of its activity in T. brucei, this association does not promote degradation of mouse ODC in the trypanosomal cells, in contrast to the results observed in mammalian cells (18) .

In Vitro Inhibition And Degradation of Mouse ODC Initiated by the Rat Antizyme Expressed in Trypanosomes

Since in vitro incubation of rat antizyme with mouse ODC can inhibit ODC activity and direct its degradation (14, 18) , we determined whether or not antizyme expressed in T. brucei could do so as well. Purified mouse ODC (0.5 unit) was mixed either with the antizyme from Tb/AZ (1.5 10cell lysate of Tb/AZ) or with the in vitro translated antizyme (approximately 0.25 unit of antizyme). The mixtures were incubated on ice for 30 min and assayed for ODC activity. As shown in Fig. 7, the antizyme from Tb/AZ was able to inhibit mouse ODC activity in vitro (compare the columns 4 and 5). The cell lysate from 1.5 10cells of Tb/AZ inhibited about 20% of the mouse ODC activity, i.e. it contained about 0.1 unit of antizyme activity. Immunostaining of the antizyme fractionated by SDS-PAGE and densitometric analysis of the antizyme-specific bands (data not shown) indicated that approximately 30-fold more immunoreactive antizyme from Tb/AZ was needed to achieve equal inhibitory effect compared with antizyme generated by in vitro translation. This suggests that antizyme from the trypanosomal cell lysate is much less active than that produced by in vitro translation.


Figure 7:In vitro inhibitory effect of rat antizyme expressed in T. brucei on mouse ODC. The purified mouse ODC ( mODC) (approximately 0.5 unit) was mixed with either rabbit reticulocyte lysate ( RRL) or about 0.25 unit of the in vitro translated antizyme (18) ( iv AZ), or lysates from 1.5 10cells of either pTSA-Hyg2 ( Tb/TSA) or pAZ ( Tb/AZ) transformants. The mixtures were then assayed for ODC activity. The endogenous trypanosomal ODC activities (0.5 unit) from Tb/TSA and Tb/AZ have already been subtracted.



We further examined the in vitro degradation of native mouse ODC catalyzed by various samples of rat antizyme. A rabbit reticulocyte extract system was used as the in vitro degradation system as before (18) . Mouse ODC was labeled with L-[S]methionine by in vitro translation. The labeled mouse ODC was mixed with (i) the in vitro translated antizyme; (ii) the trypanosomal cell lysates from Tb/AZ; (iii) the lysates from untransformed T. brucei; (iv) a combination of (i) and (ii); and (v) a combination of (i) and (iii). The mixtures were then each added to the rabbit reticulocyte lysate for the in vitro mouse ODC degradation. Fig. 8 A demonstrates that mouse ODC is degraded in the presence of the in vitro translated antizyme, as observed previously (18, 19) . However, the antizyme expressed in Tb/AZ did not promote in vitro degradation of mouse ODC. Furthermore, the mouse ODC degradation promoted by the in vitro translated antizyme was inhibited by the cell lysate of untransformed T. brucei or that of Tb/AZ (Fig. 8 A). These results suggest the presence of some inhibitory factor(s) in the trypanosomal cell lysates, which may interfere with the degradation of mouse ODC mediated by antizyme.


Figure 8:In vitro degradation of mouse ODC. Mouse ODC was labeled with [S]methionine ( *m) by in vitro translation (18), mixed with the in vitro translated antizyme ( ivAZ) and/or T. brucei cell lysates (1.5 10cells), followed by the addition of rabbit reticulocyte extract and an ATP-regenerating system (18). After incubation at 37 °C for 1 h, the mixtures were analyzed by SDS-PAGE and autoradiography. Panel A, from left to right: first lane, S-labeled mouse ODC ( *m); second lane, labeled mouse ODC plus in vitro translated antizyme ( ivAZ); third lane, mouse ODC plus the lysate of T. brucei; fourth lane, mouse ODC plus the lysate of Tb/AZ; fifth lane, mouse ODC plus in vitro translated antizyme and the lysate of T. brucei; sixth lane, mouse ODC plus in vitro translated antizyme and the lysate of Tb/AZ. Panel B, T. brucei cell lysates were centrifuged (16,000 g), and the unboiled and boiled supernatant ( s) and pellet ( p) fractions were added to the in vitro degradation system and analyzed by SDS-PAGE and autoradiography.



To characterize further the property of the degradation inhibitory factor(s), sonicated trypanosome cell lysate was centrifuged at 16,000 g for 30 min. The supernatant and the pellet after resuspension were added to the above mentioned in vitro degradation system, or both fractions were boiled for 5 min before being added to the in vitro degradation system. The results in Fig. 8B indicated that this inhibitory factor(s) is in the soluble fraction and heat-labile.

Identification of Proteasomes in T. brucei

Mouse ODC is degraded by the 26 S proteasome in the presence of antizyme in vitro (19, 41, 42) . We therefore determined whether proteasomes are present in trypanosomal cells. Trypanosomal cells and rabbit blood cells were each fractionated by a method used previously for mammalian proteasome purification, essentially as described (35) . The resulting preparations were each subjected to electrophoresis on a native gel, and the protease activity was detected by overlaying the gel with a mixture of the fluorogenic peptides Pro-Phe-Arg-MCA and Suc-Leu-Leu-Val-Tyr-MCA. The expected two bands were present in the mammalian derived preparation, corresponding to the 26 S and 20 S proteasomes (Fig. 9 A). The corresponding preparation from T. brucei also showed two distinct bands of activity, but both bands migrated faster than the rabbit 26 S and 20 S proteasomes. One of these two bands (see band a in Fig. 9 A) migrated slightly faster than the rabbit 20 S proteasome, and there is a protein band observed on the Coomassie Blue-stained gel corresponding to this activity (Fig. 9 B). However, the major proteolytic activity ( band b in Fig. 9 A) from T. brucei migrated much faster than the others. We then excised the 20 S proteasome band of rabbit and activity bands a and b of trypanosome from the nondenaturing gel. They were subjected to SDS-PAGE and immunostained with the antibodies against the human 20 S proteasome (35) . As shown in Fig. 9 C, the antibodies cross-reacted with the proteins of activity band a from T. brucei by reacting strongly with a protein band at 48 kDa and somewhat weakly with two other protein bands at 51 and 106 kDa, respectively. These sizes are much larger than those from the rabbit 20 S proteasome (24-32 kDa) recognized by the same antibodies (35) . It is yet to be determined whether these T. brucei proteins on the immunostaining blot represent the genuine subunits of the proteasome in trypanosome. On the other hand, no significant cross-reacting signal was observed from the activity band b on the same immunoblot, suggesting that the proteins in this activity band are sufficiently distinct from those in the mammalian 20 S proteasome. It is presently unclear whether the activity band b represent a distinctive form of T. brucei proteasome or a contaminating protease activity cosedimented at 100,000 g with the proteasome. We thus conclude that a proteasome is present in T. brucei. It has properties distinctive from those of the mammalian proteasomes, which may constitute part of the reasons why mouse ODC cannot be degraded inside T. brucei even in the presence of rat antizyme.


Figure 9: Identification of proteasome in T. brucei. Proteasomes were isolated from rabbit blood cells ( Rb) and T. brucei cells ( Tb) as described under ``Experimental Procedures.'' The proteasome preparations were separated on a native gel, and the protease activities were detected with a mixture of fluorogenic peptides Suc-Leu-Leu-Val-Tyr-MCA and Pro-Phe-Arg-MCA ( panel A). The same gel was stained with Coomassie Brilliant Blue ( panel B). Or, the activity bands (20 S of rabbit, and a and b of T. brucei) were excised and subjected to SDS-PAGE and immunostaining with antibodies against the human 20 S proteasome ( panel C).




DISCUSSION

The activity and stability of ODC in many living organisms, like many other enzymes of biosynthetic pathways, are regulated by the end products of its associated metabolic pathway. ODC activity varies inversely with the supply of polyamines among all the eukaryotes so far tested, including mammals (9) , Xenopus laevis (36) , Neurospora crassa (38) , Saccharomyces cerevisiae (37) , and Pharsarum polycepharum (43) . The mechanism of inactivation of ODC varies in different organisms. In Physarum, polyamines induce the transition from active (A-form) to inactive (B-form) states (43) . Studies of S. cerevisiae (37) suggest that polyamines regulate ODC expression at a post-translational step prior to assembly of the active form of the enzyme. In the fungus N. crassa, polyamines appear to repress synthesis of ODC as well as stimulate its degradation (44) . In mammalian (9) and Xenopus cells (36) , inactivation of ODC is associated with the presence of the ODC antizyme. ODC antizyme is defined as the inhibitory protein whose synthesis is induced by the products of ODC, the polyamines (9) . Antizyme inhibits ODC activity and mediates the degradation of ODC protein in the vertebrates (18, 19) . We have examined the effects of exogenous polyamines on the ODC activity in T. brucei. Our results indicated that the trypanosomal ODC activity in the procyclic form is unaffected by the presence of putrescine and spermidine in the culture medium, contrary to the results in eukaryotic cells examined previously. In view of the fact that polyamines can effectively enter T. brucei cells (7, 39) , our results suggest that the machinery for polyamine-dictated regulation of ODC activity and stability may not exist in T. brucei.

The stable activity and protein level of trypanosomal ODC indicate that an antizyme homolog is absent in T. brucei, a conclusion consistent with the negative outcome of several tests applied in our present studies. In the vertebrates, antizyme inhibits ODC activity by forming an antizyme-ODC complex. Li and Coffino (14, 30) have demonstrated previously that a region near the amino terminus of mouse ODC as well as the carboxyl-terminal portion of rat antizyme are required for the in vitro association between the two proteins. Our immunoprecipitation experiments demonstrated that mouse ODC and rat antizyme are indeed associated in T. brucei cells. However, only 20-25% of mouse ODC was associated with antizyme in Tb/MOD-AZ. This percentage corresponds well with the extent of inhibition of mouse ODC activity by antizyme (also 20-25%) from the same T. brucei transformant. We estimated that both mouse ODC and rat antizyme are expressed at equal molar ratio in Tb/MOD-AZ. However, the antizyme expressed in trypanosome is much less reactive than that from in vitro translation. One simple explanation is that there may be some factor(s) in T. brucei cells partially blocking the ODC-antizyme association. However, further evidence on the presence of putative inhibitory factor(s) is currently unavailable.

Li and Coffino (18) demonstrated that antizyme binding to mouse ODC causes the exposure of the carboxyl terminus of the latter and subjects the enzyme protein to degradation in reticulocyte lysates. Our current results demonstrated that in T. brucei cells antizyme-mouse ODC association leads to inactivation of the ODC activity but not to the degradation of mouse ODC. The results from Fig. 8indicated that some soluble and heat-labile factor(s) are present in T. brucei which inhibits the degradation of mouse ODC in the reticulocyte lysates. This inhibitory factor(s) may also contribute to the inhibition of mouse ODC degradation mediated by the antizyme in Tb/MOD-AZ cells. However, the working mechanism of this inhibitory factor(s) is unclear. It is possible this inhibitory factor(s) is also the one(s) partially blocking the association between mouse ODC and antizyme in Tb/MOD-AZ cells.

It has been shown that degradation of mammalian ODC is mediated by antizyme and 26 S proteasome in reticulocyte lysates (41) . Here we also identified the presence of proteasome in T. brucei. However, the proteasome identified from T. brucei is probably different from the mammalian counterparts. Their mobility on native gels is faster than rabbit 26 S and 20 S proteasomes; presumably the complexes are smaller than 20 S. The substrate specificity analysis revealed that trypanosomal proteasome preparations demonstrated much higher trypsin-like activity than chymotrypsin-like activity, whereas the proteasome preparations from rabbit reticulocyte lysate have the opposite specificity.() Two lines of recent evidence indicated that the chymotryptic activities of proteasomes are primarily responsible for the major proteolysis of ODC. Mamroud-Kidron et al. (45) showed that in a mutant yeast cell line defective in the chymotryptic activity of proteasome, degradation of mouse ODC and yeast ODC was severely inhibited, whereas in wild type yeast cells, both proteins were rapidly degraded. Sequence analysis of peptides cleaved by the mammalian 26 S proteasome in vitro (42) revealed that chymotryptic-like activity of 26 S proteasome is predominant in the endoproteolysis of ODC, although other type of activities are also present. These differences between T. brucei and mammalian proteasomes may thus contribute to the stability of mouse ODC in T. brucei, in addition to the inhibitory factor(s) discussed above.

In addition to the antizyme-dependent degradation of ODC by 26 S proteasome in mammalian systems, it is generally believed that the 26 S proteasome performs selective degradation of eukaryotic cellular proteins through a ubiquitination pathway (23, 46) . Among the few physiological substrates identified in the ubiquitin proteolytic pathway thus far (46) , one of them is the mitotic cyclin (25) . The cyclins associate with protein kinases of the p34family to form important regulator of the cell cycle. The proteasomes may be involved in the degradation of mitotic cyclins. For example, mutations in a proteasome-related gene in yeast cause defects in nuclear division. These defects can be suppressed by deletion of a mitotic cyclin gene (47) . Recently, a mitotic cyclin gene has also been identified and isolated from T. brucei (48) . Its protein abundance increases during the mitotic phase and decreases during the S phase of the cell cycle in the procyclic form of T. brucei (48) , presumably because of degradation of this cyclin protein by the cell cycle-regulated proteolysis, namely, the proteasomes. Furthermore, certain proteins in T. brucei are known to turn over at different rates during individual phases of its life cycle. For example, the half-life of cytochrome c is approximately 1 h in the bloodstream form, but no detectable turnover of this protein occurs in the procyclic form (49) . Whether the proteasomes in the bloodstream forms differ from those in the procyclic forms by their specific modes of protein degradation could constitute an interesting subject for further exploration into possible regulations of the life cycle of T. brucei by alterations of the proteasome functions.


FOOTNOTES

*
This work is supported by a National Institutes of Health Grant AI21786 (to C. C. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Pharmaceutical Chemistry, Box 0446, University of California, San Francisco, CA 94143. Tel.: 415-476-1321; Fax: 415-476-3382.

The abbreviations used are: ODC, ornithine decarboxylase; DFMO, -DL-difluoromethylornithine; HTC, hepatoma cells; PAGE, polyacrylamide gel electrophoresis; Suc, succinyl; MCA, 4-methyl-coumaryl-7-amide.

S.-b. Hua, T. Nguyen, and C. C. Wang, unpublished data.


ACKNOWLEDGEMENTS

-We thank Dr. Jürg Sommer for providing pTOD, pMOD, and the T. brucei transformation vector, pTSA-Hyg2. We are grateful to Dr. Martin Rechsteiner for the antibody against the human 20 S proteasome, and to Dr. O. Jänne for the antiserum against mouse ODC.


REFERENCES
  1. Tabor, C. W., and Tabor, H. (1984) Annu. Rev. Biochem. 53, 749-790 [CrossRef][Medline] [Order article via Infotrieve]
  2. Oka, T., and Borellini, F. (1989) in Ornithine Decarboxylase: Biology, Enzymology, and Molecular Biology (Hayashi, S., ed) pp. 7-19, Pergamon Press, New York
  3. Jänne, J., and Alhonen-Hongisto, L. (1989) in Ornithine Decarboxylase: Biology, Enzymology, and Molecular Biology (Hayashi, S., ed) pp. 59-85, Pergamon Press, New York
  4. Bacchi, C. J., Nathan, H. C., Hutner, S. H., McCann, P. P., and Sjoerdsma, A. (1980) Science 210, 332-334 [Medline] [Order article via Infotrieve]
  5. Sjoerdsma, A., and Schechter, P. J. (1984) Clin. Pharmacol. Ther. 35, 287-300 [Medline] [Order article via Infotrieve]
  6. Bacchi, C. J., Garofalo, J., Mockenhaupt, D., McCann, P. P., Diekema, K. A., Pegg, A. E., Nathan, H. C., Mullaney, E. A., Chunosoff, L., Sjoerdsma, A., and Hutner, S. H. (1983) Mol. Biochem. Parasitol. 7, 209-225 [Medline] [Order article via Infotrieve]
  7. Nathan, H. C., Bacchi, C. J., Hutner, S. H., Rescigno, D., McCann, P. P., and Sjoerdsma, A. (1981) Biochem. Pharmacol. 30, 3010-3013 [Medline] [Order article via Infotrieve]
  8. Phillips, M. A., Coffino, P., and Wang, C. C. (1988) J. Biol. Chem. 263, 17933-17941 [Abstract/Free Full Text]
  9. Heller, J. S., Fong, W. F., and Canellakis, E. S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 1858-1862 [Abstract]
  10. Phillips, M. A., Coffino, P., and Wang, C. C. (1987) J. Biol. Chem. 262, 8721-8727 [Abstract/Free Full Text]
  11. Ghoda, L., van Daalen Wetters, T., Macrae, M., Ascherman, D., and Coffino, P. (1989) Science 243, 1493-1495 [Medline] [Order article via Infotrieve]
  12. Ghoda, L., Phillips, M. A., Bass, K. E., Wang, C. C., and Coffino, P. (1990) J. Biol. Chem. 265, 11823-11826 [Abstract/Free Full Text]
  13. Bass, K. E., Sommer, J. M., Cheng, Q. L., and Wang, C. C. (1992) J. Biol. Chem. 267, 11034-11037 [Abstract/Free Full Text]
  14. Li, X., and Coffino, P. (1992) Mol. Cell. Biol. 12, 3556-3562 [Abstract]
  15. Murakami, Y., and Hayashi, S. (1985) Biochem. J. 226, 893-896 [Medline] [Order article via Infotrieve]
  16. Murakami, Y., Matsufuji, S., Miyazaki, Y., and Hayashi, S. (1992) J. Biol. Chem. 267, 13138-13141 [Abstract/Free Full Text]
  17. Fong, W. F., Heller, J. S., and Canellakis, E. S. (1976) Biochim. Biophys. Acta 428, 456-465 [Medline] [Order article via Infotrieve]
  18. Li, X., and Coffino, P. (1993) Mol. Cell. Biol. 13, 2377-2383 [Abstract]
  19. Murakami, Y., Matsufuji, S., Kameji, T., Hayashi, S., Igarashi, K., Tamura, T., Tanaka, K., and Ichihara, A. (1992) Nature 360, 597-599 [CrossRef][Medline] [Order article via Infotrieve]
  20. Bercovich, Z., Rosenberg-Hasson, Y., Ciechanover, A., and Kahana, C. (1989) J. Biol. Chem. 264, 15949-15952 [Abstract/Free Full Text]
  21. Rosenberg-Hasson, Y., Bercovich, Z., Ciechanover, A., and Kahana, C. (1989) Eur. J. Biochem. 185, 469-474 [Abstract]
  22. Orlowski, M. (1990) Biochemistry 29, 10289-10297 [Medline] [Order article via Infotrieve]
  23. Rechsteiner, M., Hoffman, L., and Dubiel, W. (1993) J. Biol. Chem. 268, 6065-6068 [Free Full Text]
  24. Rivett, A. J. (1993) Biochem. J. 291, 1-10 [Medline] [Order article via Infotrieve]
  25. Glotzer, M., Murray, A. W., and Kirschner, M. W. (1991) Nature 349, 132-138 [CrossRef][Medline] [Order article via Infotrieve]
  26. Scheffner, M., Werness, B. A., Huibregtse, J. M., Levine, A. J., and Howley, P. M. (1990) Cell 63, 1129-1136 [Medline] [Order article via Infotrieve]
  27. Cunningham, I. (1977) J. Protozool. 24, 325-329 [Medline] [Order article via Infotrieve]
  28. Matsufuji, S., Miyazaki, Y., Kanamoto, R., Kameji, T., Murakami, Y., Baby, T. G., Fujita, K., Ohno, T., and Hayashi, S. (1990) J. Biochem. ( Tokyo) 108, 365-371 [Abstract]
  29. Miyazaki, Y., Matsufuji, S., and Hayashi, S. (1992) Gene ( Amst.) 113, 191-197 [CrossRef][Medline] [Order article via Infotrieve]
  30. Li, X., and Coffino, P. (1994) Mol. Cell. Biol. 14, 87-92 [Abstract]
  31. Sommer, J. M., Cheng, Q. L., Keller, G. A., and Wang, C. C. (1992) Mol. Biol. Cell 3, 749-759 [Abstract]
  32. Hua, S. B., and Wang, C. C. (1994) J. Cell. Biochem. 54, 20-31 [Medline] [Order article via Infotrieve]
  33. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd. ed, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  34. Harlow, E., and Lane, D. (1988) Antibody: A Laboratory Manual, pp. 421-470, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  35. Hoffman, L., Pratt, G., and Rechsteiner, M. (1992) J. Biol. Chem. 267, 22362-22368 [Abstract/Free Full Text]
  36. Baby, T. G., and Hayashi, S. (1991) Biochim. Biophys. Acta 1092, 161-164 [Medline] [Order article via Infotrieve]
  37. Fonzi, W. A. (1989) J. Biol. Chem. 264, 18110-18118 [Abstract/Free Full Text]
  38. Davis, R. H., Krasner, G. N., DiGangi, J. J., and Ristow, J. L. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4105-4109 [Abstract]
  39. Tekwani, B. L., Bacchi, C. J., and Pegg, A. E. (1992) Mol. Cell. Biochem. 117, 53-61 [Medline] [Order article via Infotrieve]
  40. Gupta, M., and Coffino, P. (1985) J. Biol. Chem. 260, 2941-2944 [Abstract]
  41. Murakami, Y., Matsufuji, S., Tanaka, K., Ichihara, A., and Hayashi, S. (1993) Biochem. J. 295, 305-308 [Medline] [Order article via Infotrieve]
  42. Tokunaga, F., Goto, T., Koide, T., Murakami, Y., Hayashi, S., Tamura, T., Tanaka, K., and Ichihara, A. (1994) J. Biol. Chem. 269, 17382-17385 [Abstract/Free Full Text]
  43. Mitchell, J. L., and Wilson, J. M. (1983) Biochem. J. 214, 345-351 [Medline] [Order article via Infotrieve]
  44. Barnett, G. R., Seyfzadeh, M., and Davis, R. H. (1988) J. Biol. Chem. 263, 10005-10008 [Abstract/Free Full Text]
  45. Mamroud-Kidron, E., Rosenberg-Hasson, Y., Rom, E., and Kahana, C. (1994) FEBS Lett. 337, 239-242 [CrossRef][Medline] [Order article via Infotrieve]
  46. Rechsteiner, M. (1991) Cell 66, 615-618 [Medline] [Order article via Infotrieve]
  47. Friedman, H., and Snyder, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2031-2035 [Abstract]
  48. Affranchino, J. L., Gonzalez, S. A., and Pays, E. (1993) Gene ( Amst.) 132, 75-82 [Medline] [Order article via Infotrieve]
  49. Torri, A. F., Bertrand, K. I., and Hajduk, S. L. (1993) Mol. Biochem. Parasitol. 57, 305-315 [Medline] [Order article via Infotrieve]

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