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1.
Parasite Immunol ; 22(12): 639-50, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11123756

ABSTRACT

A purified flagellar pocket fraction of the Trypanosoma brucei AnTat 1.1E clone was used for the generation of polyclonal antiserum in rats. Anti-flagellar pocket antibodies present in this serum recognized several proteins distinct from the major variant surface glycoprotein (VSG). In Balb/c mice, flagellar pocket immunization resulted in partial resistance towards the challenge with a low dose of parasites. This was accompanied by the induction of specific IgG2a antibodies. In an attempt to discover protective parasite antigens, antiflagellar pocket serum was used for the screening of a T. brucei bloodstream form cDNA library constructed in the lambdagt11 bacteriophage expression system. Through antibody panning and VSG elimination, 15 specific cDNA inserts were selected. Most intriguing was the observation that in addition to two clones encoding the invariant surface glycoprotein 75 (ISG75), 10 out of 15 independently selected cDNA inserts encoded the trypanosome heat shock protein 60 (tHSP60).


Subject(s)
Antibodies, Protozoan , Chaperonin 60/immunology , Flagella/immunology , Trypanosoma brucei brucei/immunology , Trypanosomiasis, African/prevention & control , Animals , Antibody Specificity , Cross Reactions , Female , Gene Expression , Gene Library , Membrane Glycoproteins/immunology , Mice , Mice, Inbred BALB C , Protozoan Proteins/immunology , Vaccination , Variant Surface Glycoproteins, Trypanosoma/immunology
2.
Gene ; 255(1): 43-50, 2000 Sep 05.
Article in English | MEDLINE | ID: mdl-10974563

ABSTRACT

During the screening of a Trypanosoma brucei brucei (T. b. brucei) cDNA library constructed from bloodstream form mRNA, we identified a 2.3kb cDNA encoding a proteasome beta subunit (ORF1) and a putative zinc finger protein (ORF2). Northern blot analysis indicated the presence of a digenic transcript as well as the two individual messengers in both procyclic and bloodstream forms of the parasite. Southern blot analysis showed the relevant locus to be unique. ORF1 encoded a 22.7kDa protein sharing over 50% identity with the eukaryotic PRCE (aka beta5) proteasome beta subunit. This protein contained a beta amino acid signature and residues involved in the catalytic activity. Further phylogenetic analysis indicated that this subunit as well as those from other kinetoplastids could be confidentially assigned to extant eukaryotic subfamilies such as beta1, beta2, and beta5. ORF2 encoded a 14.6kDa putative zinc finger protein containing five repeats of a CCHC motif commonly present in retroviral nucleocapsid proteins as well as proteins involved in vertebrate embryogenesis.


Subject(s)
Cysteine Endopeptidases/genetics , DNA, Complementary/genetics , Multienzyme Complexes/genetics , Trypanosoma brucei brucei/genetics , Zinc Fingers/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/chemistry , Molecular Sequence Data , Open Reading Frames , Phylogeny , Proteasome Endopeptidase Complex , Protein Subunits , RNA, Messenger/genetics , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transcription, Genetic
3.
Mol Microbiol ; 36(2): 328-40, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10792720

ABSTRACT

The protozoan parasite Trypanosoma brucei develops antigenic variation to escape the immune response of its host. To this end, the trypanosome genome contains multiple telomeric expression sites competent for transcription of variant surface glycoprotein genes, but as a rule only a single antigen is expressed at any time. We used reverse transcription-PCR (RT-PCR) to analyse transcription of different segments of the expression sites in different variant clones of two independent strains of T. brucei. The results indicated that RNA polymerase is installed and active at the beginning of many, if not all, expression sites simultaneously, but that a progressive arrest of RNA elongation occurs in all but one site. This defect is linked to inefficient RNA processing and RNA release from the nucleus. Therefore, functional transcription in the active site appears to depend on the selective recruitment of a RNA elongation/processing machinery.


Subject(s)
DNA-Directed RNA Polymerases/metabolism , Gene Expression Regulation , RNA, Protozoan/metabolism , Trypanosoma brucei brucei/genetics , Variant Surface Glycoproteins, Trypanosoma/genetics , Animals , Cell Nucleus/metabolism , Genetic Variation/genetics , Polymerase Chain Reaction , RNA Processing, Post-Transcriptional , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Telomere/genetics , Transcription, Genetic , Trypanosoma brucei brucei/growth & development , Variant Surface Glycoproteins, Trypanosoma/metabolism
4.
J Biol Chem ; 275(16): 12147-55, 2000 Apr 21.
Article in English | MEDLINE | ID: mdl-10766850

ABSTRACT

The glycosylphosphatidylinositol-specific phospholipase C or VSG lipase is the enzyme responsible for the cleavage of the glycosylphosphatidylinositol anchor of the variant surface glycoprotein (VSG) and concomitant release of the surface coat in Trypanosoma brucei during osmotic shock or extracellular acidic stress. In Xenopus laevis oocytes the VSG lipase was expressed as a nonacylated and a thioacylated form. This thioacylation occurred within a cluster of three cysteine residues but was not essential for catalytic activity per se. These two forms were also detected in trypanosomes and appeared to be present at roughly equivalent amounts. A reversible shift to the acylated form occurred when cells were triggered to release the VSG by either nonlytic acid stress or osmotic lysis. A wild type VSG lipase or a gene mutated in the three codons for the acylated cysteines were reinserted in the genome of a trypanosome null mutant for this gene. A comparative analysis of these revertant trypanosomes indicated that thioacylation might be involved in regulating enzyme access to the VSG substrate.


Subject(s)
Cysteine/metabolism , Trypanosoma brucei brucei/enzymology , Type C Phospholipases/metabolism , Acylation , Animals , Animals, Genetically Modified , Electrophoresis, Polyacrylamide Gel , Glycosylphosphatidylinositol Diacylglycerol-Lyase , Mutagenesis, Site-Directed , Myristic Acid/metabolism , Oocytes/metabolism , Plasmids , RNA, Messenger/metabolism , Structure-Activity Relationship , Sulfhydryl Compounds/metabolism , Transfection , Trypanosoma brucei brucei/genetics , Type C Phospholipases/genetics , Xenopus laevis
5.
Mol Biochem Parasitol ; 106(1): 109-20, 2000 Feb 25.
Article in English | MEDLINE | ID: mdl-10743615

ABSTRACT

The protozoan parasite Trypanosoma brucei relies on trans-splicing of a common spliced leader (SL) RNA to maturate mRNAs. Using the yeast two-hybrid system a protein (TSR1IP) was identified that interacts with the T. brucei serine-arginine (SR) protein termed TSR1. TSR1IP shows homology to U1 70 kDa proteins, and contains an SR rich domain as well as an acidic/arginine domain homologous to the U1 70 kDa poly(A) polymerase inhibiting domain. This protein is localized in the nucleoplasm and excluded from the nucleolus in trypanosomal bloodstream and procyclic forms. Based on structural modelling predictions and on the identification of a RNA recognition motif (RRM), it was possible to demonstrate by the yeast three-hybrid system that TSR1IP interacts with the 5' splice region of the SL RNA. All the above characteristics suggest that TSR1IP could be involved in trans-splicing.


Subject(s)
Protozoan Proteins/genetics , Saccharomyces cerevisiae Proteins , Trypanosoma brucei brucei/metabolism , Amino Acid Sequence , Animals , Blotting, Northern , Blotting, Southern , Cell Nucleus/metabolism , DNA, Protozoan/genetics , Fluorescent Antibody Technique , Humans , Immunoblotting , Molecular Sequence Data , Protozoan Proteins/biosynthesis , Protozoan Proteins/metabolism , RNA Splicing , RNA, Protozoan/genetics , RNA, Protozoan/metabolism , RNA, Spliced Leader/genetics , RNA, Spliced Leader/metabolism , RNA-Binding Proteins/metabolism , Recombinant Proteins/metabolism , Ribonucleoprotein, U1 Small Nuclear/genetics , Ribonucleoprotein, U1 Small Nuclear/metabolism , Sequence Alignment , Sequence Homology , Spliceosomes/metabolism , Trypanosoma brucei brucei/genetics
6.
J Biol Chem ; 275(6): 4072-80, 2000 Feb 11.
Article in English | MEDLINE | ID: mdl-10660566

ABSTRACT

Heterologous expression in COS cells followed by orientation-specific polymerase chain reaction to select and amplify cDNAs encoding surface proteins in Trypanosoma brucei resulted in the isolation of a cDNA ( approximately 1.4 kilobase) which encodes an acidic, alanine-rich polypeptide that is expressed only in bloodstream forms of the parasite and has been termed bloodstream stage alanine-rich protein (BARP). Analysis of the amino acid sequence predicted the presence of a typical NH(2)-terminal leader sequence as well as a COOH-terminal hydrophobic extension with the potential to be replaced by a glycosylphosphatidylinositol anchor. A search of existing protein sequences revealed partial homology between BARP and the major surface antigen of procyclic forms of Trypanosoma congolense. BARP migrated as a complex, heterogeneous series of bands on Western blots with an apparent molecular mass ( approximately 50-70 kDa) significantly higher than predicted from the amino acid sequence ( approximately 26 kDa). Confocal microscopy demonstrated that BARP was present in small discrete spots that were distributed over the entire cellular surface. Detergent extraction experiments revealed that BARP was recovered in the detergent-insoluble, glycolipid-enriched fraction. These data suggested that BARP may be sequestered in lipid rafts.


Subject(s)
Protozoan Proteins/chemistry , Trypanosoma brucei brucei/chemistry , Amino Acid Sequence , Animals , COS Cells , Cloning, Molecular , Escherichia coli , Fluorescent Antibody Technique , Glycolipids/chemistry , Membrane Proteins/chemistry , Microscopy, Confocal , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/metabolism , Restriction Mapping , Sequence Homology, Amino Acid
7.
Mol Biochem Parasitol ; 102(1): 103-15, 1999 Jul 30.
Article in English | MEDLINE | ID: mdl-10477180

ABSTRACT

The protozoan parasite Trypanosoma brucei relies on trans-splicing to process its mRNAs. A novel nuclear serine/arginine (SR)-rich trypanosomal protein (TSR1) was characterized which contains two RNA recognition motifs. The TSR1 protein appears to be homologous to RNA-binding SR proteins of the cis-splicing machinery from higher eukaryotes. Moreover, in the yeast two-hybrid system, TSR1 is able to interact with the human splicing factors involved in the recognition of the 3' splicing site (U2AF35/U2AF65). In both procyclic and bloodstream forms of T. brucei, TSR1 was found to localize in the nucleus. In the bloodstream stage TSR1 showed the speckles pattern characteristic of SR proteins involved in cis-splicing. Moreover, TSR1 was able to specifically bind the spliced leader (SL) RNA involved in trans-splicing in trypanosomes by the yeast three-hybrid system. These and other observations suggest that TSR1 may be involved in trans-splicing in T. brucei.


Subject(s)
Fungal Proteins/chemistry , Fungal Proteins/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Trans-Splicing/genetics , Trypanosoma brucei brucei/genetics , Trypanosoma brucei brucei/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA, Protozoan/analysis , Fluorescent Antibody Technique , Fungal Proteins/metabolism , Humans , Mice , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , RNA, Protozoan/metabolism , RNA, Spliced Leader/genetics , RNA-Binding Proteins/metabolism , Transcription, Genetic
8.
Cell ; 95(6): 839-46, 1998 Dec 11.
Article in English | MEDLINE | ID: mdl-9865701

ABSTRACT

Infectivity of Trypanosoma brucei rhodesiense to humans is due to its resistance to a lytic factor present in human serum. In the ETat 1 strain this character was associated with antigenic variation, since expression of the ETat 1.10 variant surface glycoprotein was required to generate resistant (R) clones. In addition, in this strain transcription of a gene termed SRA was detected in R clones only. We show that the ETat 1.10 expression site is the one selectively transcribed in R variants. This expression site contains SRA as an expression site-associated gene (ESAG) and is characterized by the deletion of several ESAGs. Transfection of SRA into T.b. brucei was sufficient to confer resistance to human serum, identifying this gene as one of those responsible for T.b. rhodesiense adaptation to humans.


Subject(s)
Genes, Protozoan , Trypanosoma brucei rhodesiense/genetics , Trypanosoma brucei rhodesiense/pathogenicity , Variant Surface Glycoproteins, Trypanosoma/genetics , Animals , Antigenic Variation , Base Sequence , Blood , DNA, Protozoan , Gene Expression , Humans , Molecular Sequence Data , Transcription, Genetic , Trypanosoma brucei rhodesiense/immunology
9.
J Biol Chem ; 272(46): 29212-21, 1997 Nov 14.
Article in English | MEDLINE | ID: mdl-9361000

ABSTRACT

A new surface membrane protein, invariant surface glycoprotein termed ISG100, was identified in Trypanosoma brucei, using catalyzed surface, radioiodination of intact cells. This integral membrane glycoprotein was purified by a combination of detergent extraction, lectin-affinity, and ion-exchange chromatography followed by preparative SDS-polyacrylamide gel electrophoresis. The protein was expressed only in bloodstream forms of the parasite, was heavily N-glycosylated, and was present in different clonal variants of the same serodeme as well as in different serodemes. The gene for this protein was isolated by screening a cDNA expression library with antibodies against the purified protein followed by screening of a genomic library. The nucleotide sequence of the gene (4050 base pairs) predicted a highly reiterative polypeptide containing three distinct domains, a unique N-terminal domain of about 10 kDa containing three potential N-glycosylation sites, which was followed by a large internal domain consisting entirely of 72 consecutive copies of a serine-rich, 17-amino acid motif (approximately 113 kDa) and terminated with an apparent transmembrane spanning region of about 3.3 kDa. The internal repeat region of this gene (3672 base pairs) represents the largest reiterative coding sequence to be fully characterized in any species of trypanosome. There was no significant homology with other known proteins, and overall the predicted protein was extremely hydrophobic. Unlike the genes for other surface proteins, the gene encoding ISG100 was present as a single copy. Although present in the flagellar pocket, ISG100 was predominantly associated with components of the pathways for endo/exocytosis, such as intracellular vesicles located in the proximity of the pocket as well a large, electron-lucent perinuclear digestive vacuole.


Subject(s)
Membrane Glycoproteins/genetics , Protozoan Proteins/genetics , Serine/chemistry , Trypanosoma brucei brucei/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA, Protozoan , Microscopy, Immunoelectron , Molecular Sequence Data , Rats , Transcription, Genetic , Trypanosoma brucei brucei/ultrastructure
11.
EMBO J ; 16(24): 7272-8, 1997 Dec 15.
Article in English | MEDLINE | ID: mdl-9405356

ABSTRACT

The Trypanosoma brucei transferrin (Tf) receptor is a heterodimer encoded by ESAG7 and ESAG6, two genes contained in the different polycistronic transcription units of the variant surface glycoprotein (VSG) gene. The sequence of ESAG7/6 differs slightly between different units, so that receptors with different affinities for Tf are expressed alternatively following transcriptional switching of VSG expression sites during antigenic variation of the parasite. Based on the sequence homology between pESAG7/6 and the N-terminal domain of VSGs, it can be predicted that the four blocks containing the major sequence differences between pESAG7 and pESAG6 form surface-exposed loops and generate the ligand-binding site. The exchange of a few amino acids in this region between pESAG6s encoded by different VSG units greatly increased the affinity for bovine Tf. Similar changes in other regions were ineffective, while mutations predicted to alter the VSG-like structure abolished the binding. Chimeric proteins containing the N-terminal dimerization domain of VSG and the C-terminal half of either pESAG7 or pESAG6, which contains the ligand-binding domain, can form heterodimers that bind Tf. Taken together, these data provided evidence that the T.brucei Tf receptor is structurally related to the N-terminal domain of the VSG and that the ligand-binding site corresponds to the exposed surface loops of the protein.


Subject(s)
Protein Structure, Secondary , Receptors, Transferrin/chemistry , Receptors, Transferrin/metabolism , Trypanosoma brucei brucei/metabolism , Variant Surface Glycoproteins, Trypanosoma/chemistry , Variant Surface Glycoproteins, Trypanosoma/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cattle , Dimerization , Female , Genes, Protozoan , Genetic Variation , Ligands , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Oocytes/physiology , Receptors, Transferrin/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Transcription, Genetic , Transferrin/metabolism , Trypanosoma brucei brucei/genetics , Variant Surface Glycoproteins, Trypanosoma/genetics , Xenopus laevis
12.
Mol Cell Biol ; 16(3): 914-24, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8622694

ABSTRACT

The polycistronic procylcin PARP (for procyclic acidic repetitive protein) A transcription unit of Trypanosoma brucei was completely characterized by the mapping of the termination region. In addition to the tandem of procyclin genes and GRESAG 2.1, this 7.5- to 9.5-kb unit contained another gene for a putative surface protein, termed PAG (for procyclin-associated gene) 3. The terminal 3-kb sequence did not contain significant open reading frames and cross-hybridized with the beginning of one or several transcription units specific to the bloodstream form. At least three separate fragments from the terminal region were able to inhibit chloramphenicol acetyltransferase expression when inserted between either the PARP, the ribosomal, or the variable surface glycoprotein promoter and a chloramphenicol acetyltransferase reporter gene. This inhibition was due to an orientation-dependent transcription termination caused by the combination of several attenuator elements with no obvious sequence conservation. The procyclin transcription terminator appeared unable to inhibit transcription by polymerase II.


Subject(s)
Membrane Glycoproteins/genetics , Protozoan Proteins , Terminator Regions, Genetic , Trypanosoma brucei brucei/metabolism , Amino Acid Sequence , Animals , Base Sequence , Molecular Sequence Data , Sequence Analysis , Transcription, Genetic , Trypanosoma brucei brucei/genetics
13.
Mol Cell Biol ; 15(10): 5598-606, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7565711

ABSTRACT

The variant surface glycoprotein (VSG) and procyclin promoters of Trypanosoma brucei recruit an RNA polymerase sharing characteristic with polymerase I, but there is no sequence homology between them nor between these promoters and ribosomal promoters. We report the detailed characterization of the VSG promoter. The 70-bp region upstream of the transcription start site was sufficient for full promoter activity. Mutational analysis revealed three short critical stretches at positions -61 to -59 (box 1), -38 to -35 (box 2), and -1 to +1 (start site), the spacing of which was essential. These elements were conserved in the promoter for a metacyclic VSG gene. Hybrid sequences containing box 1 of the VSG promoter and box 2 of the ribosomal promoter were active. A specific binding of proteins to the noncoding strand of box 2, but not to double-stranded DNA, occurred. Competition experiments indicated that these proteins also bind to the corresponding region of the metacyclic VSG, procyclin, and ribosomal promoters. Binding of such a protein, of 40 kDa, appeared to be shared by these promoters.


Subject(s)
DNA, Ribosomal/genetics , DNA-Binding Proteins/metabolism , Membrane Glycoproteins/genetics , Promoter Regions, Genetic/genetics , Trypanosoma brucei brucei/genetics , Variant Surface Glycoproteins, Trypanosoma/genetics , Animals , Base Sequence , DNA Mutational Analysis , DNA, Protozoan/genetics , DNA, Protozoan/metabolism , Molecular Sequence Data , Protozoan Proteins/metabolism , Sequence Deletion , Trypanosoma brucei brucei/growth & development
14.
EMBO J ; 14(12): 2925-34, 1995 Jun 15.
Article in English | MEDLINE | ID: mdl-7796818

ABSTRACT

The variant surface glycoprotein (VSG) and procyclin are the respective major surface antigens of the bloodstream and the procyclic forms of Trypanosoma brucei. These proteins and their mRNAs are both the most abundant and absolutely characteristic of their respective life cycle stages. We show that the 3'-terminal region of these mRNAs regulates expression of a reporter gene in an inverse manner, depending on the developmental form of the parasite. In the case of VSG mRNA, the 97 nt sequence upstream from the polyadenylation site is responsible for these effects. The regulation occurs through a variation of mRNA abundance which is not due to a change in primary transcription. In the bloodstream form this effect is manifested by an increase in RNA stability, whereas in the procyclic form it seems to be related to a reduction in the efficiency of mRNA maturation. The 3'-end of VSG mRNA can obviate the 5- to 10-fold stimulation of transcription driven by the procyclin promoter during differentiation from the bloodstream to the procyclic form. The predominance of posttranscriptional over transcriptional controls is probably linked to the organization of the trypanosome genome in polycistronic transcription units.


Subject(s)
Gene Expression Regulation, Developmental/genetics , Membrane Glycoproteins/genetics , Protozoan Proteins , RNA, Messenger/genetics , Trypanosoma brucei brucei/genetics , Variant Surface Glycoproteins, Trypanosoma/genetics , Animals , Base Sequence , Genes, Reporter/genetics , Molecular Sequence Data , Promoter Regions, Genetic/genetics , RNA, Messenger/metabolism , RNA, Protozoan/genetics , RNA, Protozoan/metabolism , Regulatory Sequences, Nucleic Acid/genetics , Transcription, Genetic , Trypanosoma brucei brucei/growth & development
15.
Mutat Res ; 342(3-4): 147-56, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7715616

ABSTRACT

Our previous cytogenetic biomonitoring of a group of inhabitants in a village (Mellery, Belgium) where exposure to a mixture of toxic environmental pollutants, (probably originating from a neighbouring chemical waste disposal site) was suspected, showed that difference in the SCE and HFC bioassays was more pronounced for children. The results of follow-up study in 1992 confirmed this surprising conclusion by an even higher incidence. As very few studies have been performed on the levels of children's biomarkers, this group of exposed populations needed to be explored further. Do children residing in the vicinity of hazardous waste sites indeed represent a population at higher risk? In the present study, we compare the performance of various bioassays (SCE, HFC, SSB and MN) in extended exposed and reference children's groups. Simultaneously, in the exposed group, we followed variation in the lymphocyte SCE frequencies as a function of time. Reversibility of the latter biomarker was ascertained subsequent to a preliminary technical remediation of the disposal site. We compared these data with those obtained from a synchronous cross-sectional study on a group of children living near a similar chemical disposal site. The two exposed populations did not differ from the reference population regarding to the SCE and HFC mean levels. Comparisons of the mean levels of the two other biomarkers, SSB and MN, showed no difference between the Mellery exposed children and the reference group from Wavre whereas significant differences appeared when the Hensies group is compared either to the Mellery or to the Wavre reference group.


Subject(s)
Environmental Pollutants/toxicity , Adolescent , Belgium , Biomarkers , Child , Child, Preschool , DNA Damage , Female , Follow-Up Studies , Humans , Longitudinal Studies , Male , Micronucleus Tests , Sister Chromatid Exchange
16.
Mutat Res ; 319(4): 317-23, 1993 Dec.
Article in English | MEDLINE | ID: mdl-7504206

ABSTRACT

By analogy to the techniques applied for monitoring biological effects of exposure to genotoxic agents in occupational populations, we have carried out cytogenetic monitoring in a group of inhabitants of a village (Mellery, Belgium) suspected to have been exposed to a variety of toxic environmental pollutants. These pollutants probably originated from a neighboring chemical wastes site. A group of 51 environmentally exposed and 52 reference persons (including children) were examined for the frequency of sister-chromatid exchanges (SCE) in their peripheral blood lymphocytes. The technique was further refined by using a high frequency cells (HFC) analysis. Analysis of the reference subgroups showed a significant difference between non-smoking adults and children. The influence of tobacco was clear, too. In the exposed group, no significant differences could be demonstrated between either the smokers or the non-smokers or the children. Furthermore, not only were the mean frequencies of SCE higher than in the respective reference subgroups but comparison between the two groups also showed a higher number of individuals presenting a HFC level above the background in the exposed group. Surprisingly, the difference was more pronounced for the children. A follow-up of the same exposed population carried out 18 months later and after remediation of the atmospheric chemical release, the previously observed tendencies in the exposure parameter remained unmodified.


Subject(s)
Environmental Monitoring/methods , Environmental Pollutants/adverse effects , Sister Chromatid Exchange/drug effects , Adult , Belgium , Cells, Cultured , Child , Female , Humans , Lymphocytes/ultrastructure , Male , Reference Values
17.
Nucleic Acids Res ; 19(21): 5857-62, 1991 Nov 11.
Article in English | MEDLINE | ID: mdl-1945875

ABSTRACT

The actin genes of Trypanosoma brucei are transcribed constitutively during the parasite life-cycle, by a polymerase sensitive to alpha-amanitin. The start region of the actin gene transcription unit was mapped by virtue of the accumulation of promoter-proximal transcripts which occurs following moderate UV irradiation. This region, located about 4 kilobases upstream from the genes, was able to direct transient expression of the bacterial Chloramphenicol Acetyl Transferase (CAT) gene in both bloodstream and procyclic forms of the parasite. The essential region of the promoter was defined by deletion, and appeared to be within 600 bp upstream from the putative transcription start site. It does not share significant homology with the other trypanosome promoters described so far (VSG, procyclin, rDNA), which all direct alpha-amanitin resistant transcription.


Subject(s)
Actins/genetics , Promoter Regions, Genetic/genetics , Trypanosoma brucei brucei/genetics , Amanitins/pharmacology , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Chloramphenicol O-Acetyltransferase/genetics , Chromosome Deletion , Cloning, Molecular , DNA Mutational Analysis , Electric Stimulation , Kinetics , Molecular Sequence Data , Plasmids/genetics , Transcription, Genetic/drug effects , Transcription, Genetic/physiology , Trypanosoma brucei brucei/growth & development
18.
Mol Cell Biol ; 11(3): 1473-9, 1991 Mar.
Article in English | MEDLINE | ID: mdl-1996104

ABSTRACT

The genes for the variant surface glycoprotein (VSG) and procyclin are expressed in a mutually exclusive manner during the life cycle of Trypanosoma brucei and synthesize the most abundant mRNAs specific to the bloodstream and procyclic stages of the parasite, respectively. Genes belonging to the polycistronic transcription unit of the VSG gene (expression site-associated genes [ESAGs]) are uniquely expressed in the bloodstream form, but some members of ESAG families (genes related to ESAGs [GRESAGs]) are independently transcribed outside the VSG gene expression site. We report here that a gene related to ESAG 2, GRESAG 2.1, is present and expressed in a procyclin gene transcription unit (PARP A locus), which is polycistronic. Members of the ESAG 2 family are thus present in the two major differentially stage-regulated transcription units of this parasite.


Subject(s)
Glycoproteins , Membrane Glycoproteins , Protozoan Proteins/genetics , Trypanosoma brucei brucei/genetics , Variant Surface Glycoproteins, Trypanosoma/genetics , Amanitins/pharmacology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA/genetics , Genes , Molecular Sequence Data , Multigene Family , Restriction Mapping , Sequence Homology, Nucleic Acid , Transcription, Genetic/drug effects
19.
Mol Biochem Parasitol ; 43(2): 279-88, 1990 Dec.
Article in English | MEDLINE | ID: mdl-1982555

ABSTRACT

The expression site for the variant surface glycoprotein (VSG) gene of Trypanosoma brucei contains several genes of unknown function (ESAGs, for expression site-associated genes). Among these, ESAG 4 shows homology to eukaryotic adenylate/guanylate cyclase genes, in the region encoding the presumptive enzyme catalytic domain. This gene belongs to a family of related sequences, and hybridizes to the genomic DNA of other trypanosomatids, such as Trypanosoma congolense, Trypanosoma vivax and Trypanosoma mega. While ESAG 4 is transcribed only in bloodstream forms by a RNA polymerase resistant to alpha-amanitin, at least three other members of this family are transcribed in both bloodstream and procyclic forms, by a RNA polymerase sensitive to the drug. These genes encode different putative transmembrane proteins showing high sequence conservation in the region corresponding to the adenylate/guanylate cyclase catalytic domain.


Subject(s)
Adenylyl Cyclases/genetics , Gene Expression Regulation, Enzymologic , Guanylate Cyclase/genetics , Multigene Family , Trypanosoma brucei brucei/genetics , Adenylyl Cyclases/biosynthesis , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Guanylate Cyclase/biosynthesis , Molecular Sequence Data , Restriction Mapping , Sequence Homology, Nucleic Acid , Transcription, Genetic , Trypanosoma brucei brucei/enzymology , Variant Surface Glycoproteins, Trypanosoma/genetics
20.
EMBO J ; 9(10): 3145-51, 1990 Oct.
Article in English | MEDLINE | ID: mdl-1698609

ABSTRACT

The variant surface glycoprotein (VSG) and procyclin are the major surface proteins of the bloodstream and procyclic stages, respectively, of Trypanosoma brucei. The promoter regions of the VSG and procyclin gene transcription units could be mapped thanks to the specific enrichment of initial transcripts that occurs following UV irradiation. Whereas the VSG gene is 45 kb distant from its promoter, procyclin genes are located immediately downstream. We show, by run-on assays on isolated nuclei and by cDNA analysis, that transcription occurs from both promoters in bloodstream as well as in procyclic forms. It is inferred that the control of the stage-specific expression of VSG and procyclin genes is not effected at the level of transcription initiation, but most probably by interfering with the elongation and stability of the specific transcripts.


Subject(s)
Membrane Glycoproteins , Promoter Regions, Genetic , Protozoan Proteins , Transcription, Genetic , Trypanosoma brucei brucei/genetics , Variant Surface Glycoproteins, Trypanosoma/genetics , Animals , Base Sequence , DNA/genetics , DNA/isolation & purification , Genes , Molecular Sequence Data , RNA/genetics , RNA/isolation & purification , Restriction Mapping , Trypanosoma brucei brucei/immunology
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