Differential regulation of two myo-inositol transporter genes of Saccharomyces cerevisiae

We previously isolated two distinct Saccharomyces cerevisiae myo-inositol transporter genes, ITR1 and ITR2 (Nikawa et al., 1991). Here, we studied the regulation of their expression by measuring steady-state mRNA levels and β-galactosidase activities of lacZ fusion genes under different conditions. The results show that the expression of the two ITR genes is differently regulated: ITR1 was repressed by inositol and choline whereas ITR2 was constitutive. Deletion analysis of the ITR1 upstream region and comparison with the upstream regions of other genes involved in phospholipid synthesis indicate that the octamer sequence 5′-TTCACATG-3′ is important for the expression and inositol/choline regulation of the ITR1 gene.     

Differential regulation of three glucose transporter genes in a renal epithelial cell line

Three hexose transporter genes, the Na(+)/glucose cotransporters SGLT1 and SGLT3 (formerly SAAT1/pSGLT2) and the facilitative transporter GLUT1, are expressed in a renal epithelial cell line with proximal tubule characteristics. A number of studies have demonstrated that SGLT1 expression is coupled to the cellular differentiation state and is also negatively regulated by its substrate glucose. In the present study, we demonstrate that SGLT3 mRNA expression is relatively unaffected by conditions promoting dedifferentiation (reseeding to a subconfluent density, activation of protein kinase C) or differentiation (confluent cell density, activation of protein kinase A) nor was expression sensitive to hyperglycemic glucose levels in the medium. We further demonstrate that protein kinase A and protein kinase C exert opposing effects on GLUT1 and SGLT1 mRNA levels in polarized cell monolayers, indicating that GLUT1 mRNA is also highly regulated in polarized epithelial cells by agents affecting cell differentiation. The relatively constitutive expression of SGLT3 mRNA suggests a novel role for this low-affinity Na(+)/glucose cotransporter, to provide concentrative glucose uptake under hyperglycemic conditions where expression of high-affinity glucose cotransporter SGLT1 mRNA is significantly downregulated.     

Trypanosoma brucei has two distinct mitochondrial DNA polymerase beta enzymes

In higher eukaryotes, DNA polymerase (pol) beta resides in the nucleus and participates primarily in DNA repair. The DNA polymerase beta from the trypanosomatid Crithidia fasciculata, however, was the first mitochondrial enzyme of this type described. Upon searching the nearly completed genome data base of the related parasite Trypanosoma brucei, we discovered genes for two pol beta-like proteins. One is approximately 70% identical to the C. fasciculata pol beta and is likely the homolog of this enzyme. The other, although approximately 30% identical within the polymerase region, has unusual structural features including a short C-terminal tail and a long N-terminal extension rich in prolines, alanines, and lysines. Both proteins, when expressed recombinantly, are active as DNA polymerases and deoxyribose phosphate lyases, but their polymerase activity optima differ with respect to pH and KCl and MgCl2 concentrations. Remarkably, green fluorescent protein fusion proteins and immunofluorescence demonstrate that both are mitochondrial, but their locations with respect to the mitochondrial DNA (kinetoplast DNA network) in this organism are strikingly different.     

Differential effects of Trypanosoma brucei gambiense and Trypanosoma brucei brucei on rat macrophages

Mammalian immune responses to Trypanosoma brucei infection are important to control of the disease. In rats infected with T. brucei gambiense (Wellcome strain; WS) or T. brucei brucei (interleukin-tat 1.4 strain [ILS]), a marked increase in the number of macrophages in the spleen can be observed. However, the functional repercussions related to this expansion are not known. To help uncover the functional significance of macrophages in the context of trypanosome infection, we determined the mRNA levels of genes associated with an increase in macrophage number or macrophage function in WS- and ILS-infected rats and in cultured cells. Specifically, we assayed mRNA levels for macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and macrophage migration inhibitory factor (MIF). Upregulation of GM-CSF and MIF mRNA levels was robust in comparison with changes in M-CSF levels in ILS-infected rats. By contrast, upregulation of M-CSF was more robust in WS-infected rats. The phagocytic activity in macrophages harvested from ILS-infected rat spleens, but not WS-infected spleens, was higher than that in macrophages from uninfected rats. These results suggest that macrophages of WS-infected rats change to an immunosuppressive type. However, when WS or ILS is cocultured with spleen macrophages or HS-P cells, a cell line of rat macrophage origin, M-CSF is upregulated relative to GM-CSF and MIF in both cell types. Anemia occurs in ILS-, but not WS-infected, rats. Treatment of spleen macrophages or HS-P cells cocultured with ILS with cobalt chloride, which mimics the effects of anemia-induced hypoxia, led to downregulation of M-CSF mRNA levels, upregulation of GM-CSF and MIF, and an increase in phagocytic activity. However, the effect of cobalt chloride on spleen macrophages and HS-P cells cocultured with WS was restricted. These results suggest that anemia-induced hypoxia in ILS-infected rats stimulates the immune system and activates macrophages.     

Compositionally and functionally distinct editosomes in Trypanosoma brucei

Uridylate insertion/deletion RNA editing in Trypanosoma brucei mitochondria is catalyzed by a multiprotein complex, the approximately 20S editosome. Editosomes purified via three related tagged RNase III proteins, KREN1 (KREPB1/TbMP90), KREPB2 (TbMP67), and KREN2 (KREPB3/TbMP61), had very similar but nonidentical protein compositions, and only the tagged member of these three RNase III proteins was identified in each respective complex. Three new editosome proteins were also identified in these complexes. Each tagged complex catalyzed both precleaved insertion and deletion editing in vitro. However, KREN1 complexes cleaved deletion but not insertion editing sites in vitro, and, conversely, KREN2 complexes cleaved insertion but not deletion editing sites. These specific nuclease activities were abolished by mutations in the putative RNase III catalytic domain of the respective proteins. Thus editosomes appear to be heterogeneous in composition with KREN1 complexes catalyzing cleavage of deletion sites and KREN2 complexes cleaving insertion sites while both can catalyze the U addition, U removal, and ligation steps of editing.     

analysis of genomic rearrangements associated with two variable antigen genes of Trypanosoma brucei.

Some variable surface glycoprotein (VSG) genes of Trypanosoma brucei undergo duplication and transposition when they are expressed. We report here the cloning of cDNAs coding for two VSGs from the ILtar 1 repertoire. Analysis of the genomes of trypanosomes expressing these and other antigens shows that there is no additional copy of the sequences coding for eight VSG in expressing clones of trypanosomes, and reveals rearrangements analogous to those previously described for the gene for another VSG from this antigen repertoire. The data indicate that duplication does not accompany the expression of these VSG genes. Transposition to a specific expression site cannot be excluded, but would have to involve either a much larger segment of DNA, or movement to a region of much greater homology with the previous flanking sequences, than is observed for VSG genes that are duplicated when expressed. It is reasoned that the control of expression by coupled duplication and transposition is not sufficient to account for the selection of a single VSG gene for expression.     

Complementation of a glucose transporter mutant of Schizosaccharomyces pombe by a novel Trypanosoma brucei gene

The African trypanosome Trypanosoma brucei has a digenetic life cycle that involves the insect vector and the mammalian host. This is underscored by biochemical switches in its nutritional requirements. In the insect vector, the parasite relies on amino acid catabolism, but in the mammalian host, it derives its energy exclusively from blood glucose. Glucose transport is facilitated, and constitutes the rate-limiting step in ATP synthesis. Here, we report the cloning of a novel glucose transporter-related gene by heterologous screening of a lambdaEMBL4 genomic library of T. brucei EATRO 164 using a rat liver glucose transporter cDNA clone. Genomic analysis shows that the gene is present as a single copy within the parasite genome. The gene encodes a protein with an estimated molecular mass of 55.9 kDa, which shares only segmental homology with members of the glucose transporter superfamily. Several potential post-translational modification sites including phosphorylation, N-glycosylation, and cotranslational myristoylation sites also punctuate the sequence. It is distinguished from classical transporter proteins by the absence of putative hydrophobic membrane-spanning domains. However, this protein was capable of complementing Schizosaccharomyces pombe glucose transporter mutants. The rescued phenotype conferred the ability of the cells to grow on a broad range of sugars, both monosaccharides and disaccharides. The kinetics of glucose uptake reflected those in T. brucei. In addition to complementation in yeast, we also showed that the gene enhanced glucose uptake in cultured mammalian cells.     

Endonuclease associations with three distinct editosomes in Trypanosoma brucei

Three distinct editosomes, typified by mutually exclusive KREN1, KREN2, or KREN3 endonucleases, are essential for mitochondrial RNA editing in Trypanosoma brucei. The three editosomes differ in substrate endoribonucleolytic cleavage specificity, which may reflect the vast number of editing sites that need insertion or deletion of uridine nucleotides (Us). Each editosome requires the single RNase III domain in each endonuclease for catalysis. Studies reported here show that the editing endonucleases do not form homodimeric domains, and may therefore function as intermolecular heterodimers, perhaps with KREPB4 and/or KREPB5. Editosomes isolated via TAP tag fused to KREPB6, KREPB7, or KREPB8 have a common set of 12 proteins. In addition, KREN3 is only found in KREPB6 editosomes, KREN2 is only found in KREPB7 editosomes, and KREN1 is only found in KREPB8 editosomes. These are the same associations previously found in editosomes isolated via the TAP-tagged endonucleases KREN1, KREN2, or KREN3. Furthermore, TAP-tagged KREPB6, KREPB7, and KREPB8 complexes isolated from cells in which expression of their respective endonuclease were knocked down were disrupted and lacked the heterotrimeric insertion subcomplex (KRET2, KREPA1, and KREL2). These results and published data suggest that KREPB6, KREPB7, and KREPB8 associate with the deletion subcomplex, whereas the KREN1, KREN2, and KREN3 endonucleases associate with the insertion subcomplex.     

Distinct roles for two RAD51-related genes in Trypanosoma brucei antigenic variation

In Trypanosoma brucei, DNA recombination is crucial in antigenic variation, a strategy for evading the mammalian host immune system found in a wide variety of pathogens. T.brucei has the capacity to encode >1000 antigenically distinct variant surface glycoproteins (VSGs). By ensuring that only one VSG is expressed on the cell surface at one time, and by periodically switching the VSG gene that is expressed, T.brucei can evade immune killing for prolonged periods. Much of VSG switching appears to rely on a widely conserved DNA repair pathway called homologous recombination, driven by RAD51. Here, we demonstrate that T.brucei encodes a further five RAD51-related proteins, more than has been identified in other single-celled eukaryotes to date. We have investigated the roles of two of the RAD51-related proteins in T.brucei, and show that they contribute to DNA repair, homologous recombination and RAD51 function in the cell. Surprisingly, however, only one of the two proteins contributes to VSG switching, suggesting that the family of diverged RAD51 proteins present in T.brucei have assumed specialized functions in homologous recombination, analogous to related proteins in metazoan eukaryotes.     

Differential effects of interferon-gamma on production of trypanosome-derived lymphocyte-triggering factor by Trypanosoma brucei gambiense and Trypanosoma brucei brucei

Trypanosome-derived lymphocyte-triggering factor (TLTF) produced by Trypanosoma brucei brucei stimulates production of interferon-gamma (IFN-gamma) by CD8+ T cells, and it is reported that, in turn, IFN-gamma stimulates proliferation of T. b. brucei. We studied the role of TLTF in trypanosome proliferation using the Wellcome strain (WS) of Trypanosoma brucei gambiense and the ILtat 1.4 strain (IL) of T. b. brucei. Increase in the number of WS in infected rats is more rapid than IL and corresponds with comparatively higher levels of IFN-gamma. Production of IFN-gamma, as measured by protein and messenger RNA (mRNA) levels, was maintained by splenocytes from WS-infected rats, whereas levels decreased in IL-infected rats, accompanied by prolongation of infection. Expression of TLTF mRNA by in vitro-cultured WS was promoted in a dose-dependent fashion by addition of recombinant rat IFN-gamma at all concentrations tested. The addition of lower concentrations of IFN-gamma to cultured IL increased expression of TLTF mRNA, whereas, in contrast to WS, addition of 100 and 1,000 U/ml IFN-gamma decreased expression of TLTF by IL. These results show that unlike WS, elevated IFN-gamma concentrations lead to decreased TLTF production by IL. It is believed that decreased TLTF production in IL-infected rats leads to lowered IFN-gamma production, thereby slowing IL proliferation.     

Presence of a lipophosphoglycan in two variants of Trypanosoma brucei brucei

For the family of Trypanosomatidae (Trypanosoma and Leishmania) the organization of the glycoproteins on the cell surface is a well documented structural feature, because their plasma membranes are potential target for chemotherapy. By using metabolic labeling ( [32P] phosphate, [3H]-myristic acid, [3H]-galactose) and by appropriate fractionated extraction, we have found a trypanosomal molecule which has electrophoretic and chromatographic properties consistent with the lipophosphoglycan of Leishmania donovani defined by Turco et al (1987) Biochemistry 26, 6233-6238 (1). In addition, the trypanosomal lipophosphoglycan, appears to have chromatographic behaviour similar to the glycolipid C of Trypanosoma brucei brucei described by Krakow et al (1986) J. Biol. Chem. 261, 12147-12153 (2). Our results suggest that the role of the trypanosomal lipophosphoglycan may take place in the orientation of the glycoproteins in the surface coat and/or corresponds to the glycolipid precursor for the anchor of variant surface glycoprotein.     

Differential regulation of glucose transporter gene expression in adipose tissue or septic rats.

To understand the molecular mechanisms responsible for the sepsis-induced enhanced glucose uptake, we have examined the levels of GLUT4 and GLUT1 mRNA and protein in the adipose tissue of septic animals. Rats were challenged with a nonlethal septic insult where euglycemia was maintained and hexose uptake in adipose tissue was markedly elevated. Northern blot analysis of total RNA isolated from epididymal fat pads indicated differential regulation of the mRNA content for the two transporters: GLUT1 mRNA was increased 2.6 to 4.6-fold, while GLUT4 mRNA was decreased by 2.5 to 2.9-fold. Despite the difference in mRNA levels, both GLUT1 and GLUT4 protein were down regulated in plasma membranes (40% and 25%, respectively) and microsomal membranes (42% and 25%, respectively) of the septic animals. The increased glucose uptake cannot be explained by the membrane content of GLUT1 and GLUT4 protein. Thus, during hypermetabolic sepsis, increased glucose utilization by adipose tissue is dependent on alternative processes.