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F Bringaud

Publications and source records attributed to F Bringaud.

31 records · Page 2Linked to original sources
PubMed1997

Mitochondrial glutamate dehydrogenase from Leishmania tarentolae is a guide RNA-binding protein.

To identify specific proteins interacting with guide RNAs (gRNAs) in mitochondrial ribonucleoprotein complexes from Leishmania tarentolae, fractionated and unfractionated mitochondrial extracts were subjected to UV cross-linking with added labeled gRNA and also with [alpha-32P]UTP-labeled endogenous RNA. An abundant 110-kDa protein (p110) localized in the T-V complex, which sediments in glycerol gradients at the leading edge of the 10S terminal uridylyltransferase peak, was found to interact with both types of labeled RNAs. The p110 protein was gel isolated and subjected to microsequence analysis, and the gene was cloned. The sequence revealed significant similarity with mitochondrial glutamate dehydrogenases. A polyclonal antiserum was raised against a recombinant fragment of the p110 gene and was used to demonstrate a stable and specific gRNA-binding activity by coimmunoprecipitation and competitive gel shift analyses. Complex formation was strongly inhibited by competition with poly(U) or by deletion or substitution of the gRNA 3' oligo(U) tail. Also, addition of a 3' oligo(U) tail to an unrelated transcript was sufficient for p110 binding. Both the gRNA-binding activity of the p110 protein and in vitro gRNA-independent and gRNA-dependent uridine insertion activities in the mitochondrial extract were inhibited by high concentrations of dinucleotides.

Amino Acid Sequence↗
PubMed1995

Functional expression and characterization of the Trypanosoma brucei procyclic glucose transporter, THT2.

The gene encoding THT2, one of two hexose-transporter isoforms present in Trypanosoma brucei, has been expressed in both Xenopus laevis oocytes and a stably transfected line of Chinese hamster ovary (CHO) cells. The heterologously expressed gene encodes a protein with pharmacological and kinetic parameters similar to those of the hexose transporter measured in procyclic-culture-form trypanosomes. The substrate recognition of the THT2 transporter differed from that of the THT1 isoform, which is expressed only in bloodstream forms, in that: (i) it has a relatively high affinity for substrate with a Km of 59 microM for 2-deoxy-D-glucose (2-DOG) and a similar high affinity for D-glucose (compared with Km of 0.5 mM for 2-DOG in bloodstream forms); (ii) the affinity for 6-deoxy-D-glucose (6-DOG) is two orders of magnitude lower than that for D-glucose, whereas the bloodstream-form transporter recognizes D-glucose and its 6-DOG analogue with similar affinity; (iii) the bloodstream-form transporter, but not THT2, recognizes 3-fluoro-3-deoxy-D-glucose. D-Fructose-transport capacity and insensitivity to D-galactose was also found in THT2-expressing CHO cells and procyclic trypanosomes. We conclude from these cumulative results that the THT2 gene encodes the transporter responsible for hexose transport in procyclic trypanosomes. The transport of 2-DOG in procyclic organisms was inhibited by both the protonophore, carbonyl cyanide 4-trifluoromethoxy phenylhydrazone (FCCP), and KCN, suggesting a requirement for a protonmotive force. However, sensitivity to these reagents depended on the external substrate concentration, with uptake being unaffected at substrate concentrations higher than 2 mM. THT2 expressed in CHO cells behaved as a facilitated transporter, and was unaffected by FCCP or KCN over the whole substrate concentration range tested.

Animals↗
PubMed1995

Characterization of two nuclear-encoded protein components of mitochondrial ribonucleoprotein complexes from Leishmania tarentolae.

Two mitochondrial proteins with molecular masses of 18 and 51 kDa were isolated from Leishmania tarentolae, and N-terminal amino-acid sequences were obtained. The cDNAs and genes encoding these proteins were cloned using RT-PCR. The proteins were identified as components of the previously characterized mitochondrial ribonucleoprotein complexes, T-Ia and T-VI, by comigration in native gels. The p18 and p51 genes contain 17 and 9-amino-acid N-terminal sequences, which are not present in the mature proteins and may represent cleavable mitochondrial targeting sequences. There are two identical p18 genes separated by 1.7 kb in tandem array and both are transcribed. The p18 amino-acid sequence is not similar to any sequence in the database. Antiserum to p18 expressed in Escherichia coli reacts with the entire tubular mitochondrion. The p51 gene is single copy, and the amino-acid sequence is similar to mitochondrial aldehyde dehydrogenases from other organisms. The N-terminal amino-acid sequences of 71 and 62-kDa mitochondrial proteins which co-migrated in native gels with several other T-complexes were also obtained. The p71 sequence proved to be similar to hsp70 sequences from other organisms. The p62 sequence was identical to an hsp60 sequence from Trypanosoma brucei.

Amino Acid Sequence↗
PubMed1995

RNA-protein interactions in the ribonucleoprotein T-complexes in a mitochondrial extract from Leishmania tarentolae.

We have investigated protein-RNA interactions and the incorporation of [alpha-32P]UTP into the guide RNA and mRNA components of the 'T-complexes' in a mitochondrial extract from Leishmania tarentolae. The terminal uridylyl transferase-containing complex T-IV is probably involved in the maturation of the 3'-oligo(U) tail of the gRNAs, but the biological function and biochemical nature of the remaining T-complexes is not known. We have found that the relative extent of labeling of the RNA components is dependent on the UTP concentration: at low levels, the main endogenous RNA components labeled are the gRNAs in T-IV; at higher levels, the mRNAs in all of the T-complexes are preferentially labeled. We also show a tentative correlation in the migration pattern of UTP-labeled T-complexes and complexes which bind exogenous labeled RNA. The relative extent of binding to specific complexes is dependent upon the type of RNA. Most of the interactions between the labeled RNAs and proteins can be disrupted by heparin or a large excess of rRNA, but two labeled complexes were resistant to competition. Most of the binding of labeled exogenous gRNA is disrupted by competition with a large excess of rRNA, but predigestion of the extract with micrococcal nuclease and saturation with rRNA uncovered a high affinity complex, which involves at least two proteins interacting with the bound gRNAs. A knowledge of the RNA and protein components may aid in understanding the biological roles of these RNP complexes.

Animals↗
PubMed1994

Characterization of glucose transport and cloning of a hexose transporter gene in Trypanosoma cruzi.

A gene from Trypanosoma cruzi, TcrHT1, which encodes a member of the glucose transporter superfamily has been cloned. The gene is similar in sequence to the T. brucei hexose transporter THT1 and the Leishmania transporter Pro-1 and is present in the T. cruzi genome as a cluster of at least eight tandemly reiterated copies. Northern blot analysis revealed two mRNA transcripts which differ in size with respect to their 3' untranslated regions. When injected with in vitro transcribed TcrHT1 mRNA, Xenopus oocytes express a hexose transporter with properties similar to those of T. cruzi. Glucose transport in T. cruzi is mediated via a carrier with unique properties when compared with the other glucose transporters already characterized among the Kinetoplastida. It is a facilitated transporter with a high affinity for D-glucose (Km = 84.1 +/- 7.9 microM and Vmax = 46 +/- 9.4 nmol/min per mg of protein) that shares with other kinetoplastid hexose transporters the ability to recognize D-fructose, which distinguishes these carriers from the human erythrocyte glucose transporter GLUT1.

Amino Acid Sequence↗
PubMed1994

Characterization of two classes of ribonucleoprotein complexes possibly involved in RNA editing from Leishmania tarentolae mitochondria.

The molecular mechanism of RNA editing in trypanosomatid mitochondria is an unsolved problem. We show that two classes of ribonucleoprotein complexes exist in a mitochondrial extract from Leishmania tarentolae and appear to be involved in RNA editing. The 'G' class of RNP complexes consists of 170-300 A particles which contain guide RNAs and proteins, show little terminal uridylyl transferase (TUTase) activity and exhibit an in vitro RNA editing-like activity. The 'T' class consists of approximately six RNP complexes, the endogenous RNA of which can be self-labeled with [alpha-32P]UTP. The most abundant T complex, T-IV, is visualized by electron microscopy as 80-140 A particles. This complex exhibits TUTase activity in the native gel and contains guide RNAs. Both G and T complexes are possibly involved with RNA editing in vivo. These results are a starting point for the analysis of the biochemistry of RNA editing.

Animals↗
PubMed1994

African trypanosome glucose transporter genes: organization and evolution of a multigene family.

Trypanosoma brucei brucei (EATRO-164) contains a tandem array of six genes encoding a glucose transporter, THT1 (trypanosome hexose transporter), followed by five genes encoding a second isoform, THT2. Two distinct clusters containing THT1 and THT2 genes have been identified in the EATRO-164 clone and in most other African trypanosome clones analyzed. Analysis of progeny from crosses between clones of T. b. brucei displaying polymorphism in THT1 copy number per cluster suggests that the two clusters of THT genes are present on homologous chromosomes. In addition, analysis of 30 African trypanosome clones revealed a high degree of polymorphism in THT1 copy number per cluster. Sequence comparison of five THT1 and two and one-half THT2 unit repeats, present within a 20-kb region, provided information about the genesis and evolution of the THT multigene family. The most divergent regions between THT1 and THT2 unit repeats probably arose from insertion of DNA fragments into an ancestral THT region. Genes of each of the different families are almost identical, and there are large regions of identity shared between THT1 and THT2 members. A mosaic copy containing most of a THT1 gene with the 3' extremity of a THT2 gene is found within the cluster. These results suggest that THT1 and THT2 arose by modification (insertion, mutation, or conversion) of duplicated ancestral genes. Functional constraints and homologous recombination may be evoked to explain the maintenance of the conserved sequences of THT1 and THT2.

Amino Acid Sequence↗
PubMed1993

Differential regulation of two distinct families of glucose transporter genes in Trypanosoma brucei.

A tandemly arranged multigene family encoding putative hexose transporters in Trypanosoma brucei has been characterized. It is composed of two 80% homologous groups of genes called THT1 (six copies) and THT2 (five copies). When Xenopus oocytes are microinjected with in vitro-transcribed RNA from a THT1 gene, they express a glucose transporter with properties similar to those of the trypanosome bloodstream-form protein(s). This THT1-encoded transport system for glucose differs from the human erythrocyte-type glucose transporter by its moderate sensitivity to cytochalasin B and its capacity to transport D-fructose. These properties suggest that the trypanosomal transporter may be a good target for antitrypanosomal drugs. mRNA analysis revealed that expression of these genes was life cycle stage dependent. Bloodstream forms express 40-fold more THT1 than THT2. In contrast, procyclic trypanosomes express no detectable THT1 but demonstrate glucose-dependent expression of THT2.

Amino Acid Sequence↗
PubMed1992

A potential hexose transporter gene expressed predominantly in the bloodstream form of Trypanosoma brucei.

A cDNA cloned from Trypanosoma brucei brucei codes for a putative membrane protein which is homologous to the erythrocyte glucose transporter and several other sugar transporters from Escherichia coli, yeast, algae and Leishmania. This cDNA hybridizes to a 2.3-kb mRNA that accumulates to a much higher degree in the bloodstream mammalian form than in the procyclic insect form of the parasite. The correlation between the expression of this gene and the hexose metabolism of Leishmania enriettii and T. brucei suggest that these 2 related genes probably encode hexose transporters. The gene encoding this mRNA is a member of a multigene family. The putative hexose transporter gene is highly conserved among Kinetoplastidae, indicating an important role for this protein in the parasite life cycle.

Amino Acid Sequence↗
PubMed1991

Exposed epitopes on a Trypanosoma equiperdum variant surface glycoprotein altered by point mutations.

African trypanosomes are covered by a dense protein layer that is immunologically distinct on different trypanosome isolates and is termed the variant surface glycoprotein (VSG). The different VSGs are expressed in a general order, where some VSGs appear preferentially early in infection and others only later. The exposed epitopes on a late antigen, VSG 78, of T.equiperdum were studied by the technique of monoclonal antibody (MAb) escape selection. MAbs that neutralize trypanosomes bearing VSG 78 reacted with the VSG only when it was attached to the trypanosome surface, suggesting that the most immunogenic surface epitopes are conformational. Trypanosome clones resistant to one of the MAbs yet still expressing VSG 78 or 78(20) were isolated in vitro. Two independent variants resistant to MAb H3 changed Ser192 to Arg by a single base change in the VSG gene and a variant resistant to MAb H21 had a single base change that converted Gln172 to Glu. A variant resistant to MAb H7 had several changes in the VSG gene, a gene conversion in the 5' region and an isolated mutation in codon 220 that is proposed to be responsible for the resistance phenotype. The isotypic bias of the MAbs against VSG 78 and an analysis of the natural variants that are resistant to MAb 78H21 suggest that glycosylation plays a role in the immunogenicity of these proteins. The analysis defines some of the exposed amino acid residues and demonstrates that VSG genes are altered by mutations and small gene conversions as well as replaced by large gene conversion-like events. The results provide biological data supporting the model of VSG structure obtained by crystallographic studies.

Amino Acid Sequence↗
PubMed1989

Active late-appearing variable surface antigen genes in Trypanosoma equiperdum are constructed entirely from pseudogenes.

The expression of genes coding for variable surface glycoproteins (VSGs) in Trypanosoma equiperdum is linked to duplicative transpositions of silent, basic copy sequences into telomere-linked expression sites. Examination of three independently derived late-appearing trypanosome clones expressing VSG-78 revealed that the expressed gene in all cases is composed of sequences derived from three or four individual silent genes. The 182 base pairs at the 3' end of the coding sequence are derived from one silent gene, the 3' donor. The remaining 5' segment is a mosaic structure containing variable-length segments derived from two, or perhaps three, related silent genes. All of the silent genes that participate in the construction of the VSG-78 expression-linked copy (ELC) genes contain multiple stop codons and are unable to code for VSGs. Individual silent pseudogenes complement one another in the mosaic structure of the 5' portions of the ELC genes and create functional VSG genes. The joining of the 3' and 5' portions of the composite genes occurs in short regions of homology and suggests a mechanism by which the ordered expression of the VSG genes is generated.

Animals↗
PubMedDate not supplied

Antigenic variation in Trypanosoma equiperdum.

Trypanosoma equiperdum is an African trypanosome that causes dourine in horses. Like the other African trypanosomes, T. equiperdum escapes elimination by the immune system of its host by using an elaborate system of antigenic variant. The trypanosomes are covered by a coat consisting of a single protein called the variable surface glycoprotein (VSG) that acts as the major trypanosome immunogen. As the host responds to one VSG, trypanosomes covered with another VSG become dominant. There is a loose order of appearance of these VSG during the infection. The factors that affect the timing of VSG expression and the effective size of the VSG repertoire in T. equiperdum are reviewed. The VSG genes are generally activated by a process of duplicative transposition involving the duplication of a silent VSG gene and inserting a copy of the gene into an expression site. The order of VSG expression is related to the amount of homology between the silent gene and the expression site. The genes expressed late in infection lack extensive homology with the expression site and depend on homology with the gene in the expression site. The genes coding for VSG expressed late in infection are hybrid genes because of this mode of transfer. This transfer mechanism allows the trypanosome to create complex VSG genes from parts of several different silent genes that are each pseudogenes. Additionally, data are presented showing that only a limited portion of the VSG is actually seen by the host immune system. These factors indicate that the effective VSG repertoire is greater than the number of VSG genes in the trypanosome genome.

Animals↗