Regulation of the ribonucleotide reductase small subunit gene by DNA-damaging agents in Dictyostelium discoideum. (33/1597)

In Escherichia coli, yeast and mammalian cells, the genes encoding ribonucleotide reductase, an essential enzyme for de novo DNA synthesis, are up-regulated in response to DNA damaging agents. We have examined the response of the rnrB gene, encoding the small subunit of ribonucleotide reductase in Dictyostelium discoideum, to DNA damaging agents. We show here that the accumulation of rnrB transcript is increased in response to methyl methane sulfonate, 4-nitroquinoline-1-oxide and irradiation with UV-light, but not to the ribonucleotide reductase inhibitor hydroxyurea. This response is rapid, transient and independent of protein synthesis. Moreover, cells from different developmental stages are able to respond to the drug in a similar fashion, regardless of the basal level of expression of the rnrB gene. We have defined the cis -acting elements of the rnrB promoter required for the response to methyl methane sulfonate and 4-nitroquinoline-1-oxide by deletion analysis. Our results indicate that there is one element, named box C, that can confer response to both drugs. Two other boxes, box A and box D, specifically conferred response to methyl methane sulfonate and 4-nitroquinoline-1-oxide, respectively.  (+info)

Faithful expression of a heterologous gene carried on an artificial macronuclear chromosome in Euplotes crassus. (34/1597)

Macronuclear chromosomes of hypotrichous ciliates are gene-sized molecules carrying the coding sequence flanked by short non-translated regions and bounded by telomeres. We have constructed artificial chromosomes for investigation of transcription in the macronucleus of Euplotes crassus. The neo gene was put under the control of the 5"-non-translated region of the TBP gene of E.crassus. These molecules were introduced into the cell with the help of liposomes. The cells were transformed and survived high concentrations of geneticin. The artificial chromosomes were kept in the macro-nucleus for at least 50 days at a copy number of about 200 per macronucleus. Expression of the gene was shown by reverse transcription of the neo messenger. The transcription start was mapped and found to coincide with that found on the natural macronuclear chromosome encoding TBP in E.crassus.  (+info)

Immunization of mice with a TolA-like surface protein of Trypanosoma cruzi generates CD4(+) T-cell-dependent parasiticidal activity. (35/1597)

The gene family encoding a trypomastigote-specific protein restricted to the part of the flagellum in contact with the cell body of the trypomastigote form of Trypanosoma cruzi has been isolated, characterized, and expressed in a baculovirus expression system. The gene family contains three tandemly repeated members that have 97 to 100% sequence identity. The predicted protein encoded by the gene family has both significant amino acid sequence identity and other physical and biological features in common with the TolA proteins of Escherichia coli and Pseudomonas aeruginosa. Based on these similarities, we have designated this gene family tolT. Immunization of mice with recombinant TolT generates a population of CD4(+) T lymphocytes that recognize T. cruzi-infected macrophages, resulting in the production of gamma interferon (IFN-gamma), which leads to NO production and a 50 to 60% reduction in parasite numbers compared to that seen with infected macrophages incubated with naive T cells. This population of T cells also produces both IFN-gamma and interleukin 2 (IL-2) but not IL-4 or IL-5 when incubated with spleen cells stimulated with TolT antigen, indicating that they are of the T-helper 1 type. T cells from mice chronically infected with T. cruzi also produce significant levels of IFN-gamma when cocultured with macrophages and either TolT protein or paraflagellar rod protein, indicating that both of these flagellar proteins produce positive T-cell responses in mice chronically infected with T. cruzi.  (+info)

Characterization of tGLP-1, a Golgi and lysosome-associated, transmembrane glycoprotein of African trypanosomes. (36/1597)

Purification of endosomal/lysosomal vesicles of Trypanosoma brucei brucei bloodstream forms and generation of monoclonal antibodies led to the isolation of antibodies directed against an 85 kDa, Golgi and endocytic traffic-associated protein termed tGLP-1, Trypanosoma Golgi/lysosome protein-1. Preliminary immunoelectron microscopical analysis revealed that the protein is present in, but not restricted to, the limiting membrane of multivesicular lysosomes and is more abundant in bloodstream forms compared to the procyclic stage. The corresponding gene was cloned and is present as a single copy. Blast searches did not reveal any homologies to other proteins and genes published. The nucleotide sequence of the gene (1848 base pairs) predicted a type 1 membrane topology with an N-terminal signal sequence (20 aa), a luminal domain with 2 N-glycosylation sites (524 aa), a transmembrane domain (23 aa), and a long cytosolic tail domain (49 aa). Polyclonal antibodies raised against the cytosolic tail confirmed the localization of the gene product to multivesicular lysosomes but revealed that the majority of the protein was in the Golgi apparatus. Colabelling with an antibody against p67, a lysosomal glycoprotein of trypanosomes, revealed extensive overlap between the proteins with opposing relative abundance. Expression of the tGLP-1 open reading frame in Leishmania resulted in Golgi localization, and in Toxoplasma, in localization to both the Golgi and endoplasmic reticulum. These data indicate conservation in the functionality of the Golgi-targeting sequence of tGLP-1.  (+info)

Control of variant surface glycoprotein gene-expression sites in Trypanosoma brucei. (37/1597)

Trypanosoma brucei has 20 similar telomeric-expression sites for variant surface glycoprotein genes. Expression sites appear to be controlled at the level of transcription initiation, resulting in only one site being active at any time. Switching between expression sites occurs at a low rate. To analyse the switching mechanism, we used trypanosomes with two expression sites tagged with two different drug-resistance genes and selected these on agarose plates containing both drugs. Double-resistant clones arose at a low frequency of 10(-7) per cell, but these behaved as if they rapidly switched between the two tagged expression sites and lost double resistance in the absence of selection. Using in situ hybridization we found that only 10% of the double-resistant cells had two fluorescent spots corresponding to transcribed expression sites. Our results suggest that: (i) a double expressor is not a stable intermediate in expression site switching; (ii) expression sites are not independently switched on and off; and (iii) expression sites can be in a 'pre-active' silent state from which they can be readily activated.  (+info)

Role of the locus and of the resistance gene on gene amplification frequency in methotrexate resistant Leishmania tarentolae. (38/1597)

The protozoan parasite Leishmania resists the antifolate methotrexate (MTX) by amplifying the R locus dihydrofolate reductase-thymidylate synthase ( dhfr-ts ) gene, the H locus ptr1 pterin reductase gene, and finally by mutation in a common folate/MTX transporter. Amplification of dhfr-ts has never been observed in Leishmania tarentolae MTX resistant mutants while ptr1 amplification is common. We have selected a L.tarentolae ptr1 null mutant for MTX resistance and observed dhfr-ts amplification in this mutant demonstrating that once a preferred resistance mechanism is unavailable, a second one will take over. By introducing the ptr1 gene at the R locus and the dhfr-ts gene at the H locus by gene targeting, we investigated the role of the resistance gene and the locus on the rate of gene amplification. Transfection studies indicated that ptr1 gave higher levels of MTX resistance than dhfr-ts. Consistent with this, when ptr1 was present as part of either the H locus or the R locus it was invariably amplified, while dhfr-ts was only amplified when ptr1 was inactivated. When dhfr-ts was present in a ptr1 null background on both the H locus and the R locus, amplification from the H locus was more frequent suggesting that both the gene and the locus are determining the frequency of gene amplification in Leishmania.  (+info)

A novel Ras-interacting protein required for chemotaxis and cyclic adenosine monophosphate signal relay in Dictyostelium. (39/1597)

We have identified a novel Ras-interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cyclic AMP (cAMP) chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced approximately 60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of the rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double-knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase, and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation.  (+info)

Inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis as antimalarial drugs. (40/1597)

A mevalonate-independent pathway of isoprenoid biosynthesis present in Plasmodium falciparum was shown to represent an effective target for chemotherapy of malaria. This pathway includes 1-deoxy-D-xylulose 5-phosphate (DOXP) as a key metabolite. The presence of two genes encoding the enzymes DOXP synthase and DOXP reductoisomerase suggests that isoprenoid biosynthesis in P. falciparum depends on the DOXP pathway. This pathway is probably located in the apicoplast. The recombinant P. falciparum DOXP reductoisomerase was inhibited by fosmidomycin and its derivative, FR-900098. Both drugs suppressed the in vitro growth of multidrug-resistant P. falciparum strains. After therapy with these drugs, mice infected with the rodent malaria parasite P. vinckei were cured.  (+info)