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Trypanosoma cruzi is the aetiologic agent of Chagas disease. Our research group studies ribosomal RNA (rRNA) gene transcription and nucleolus dynamics in this species of trypanosomes. RPA31 is an essential subunit of RNA polymerase I (Pol I) whose presence is apparently restricted to trypanosomes. Using fluorescent-tagged versions of this protein (...
Importin α, a transport factor in the classical pathway of nuclear transport of proteins in eukaryotes, has not been experimentally studied in trypanosomatids. A chimeric fluorescent version of this protein (TcImportin α-EGFP) expressed in transfected epimastigotes of Trypanosoma cruzi is characterized here. Initially, the cellular localization of...
Trypanosoma cruzi is the causative agent of American trypanosomiasis, which mainly affects populations in Latin America. Benznidazole is used to control the disease, with severe effects in patients receiving this chemotherapy. Previous studies have demonstrated the inhibition of triosephosphate isomerase from T. cruzi, but cellular enzyme inhibitio...
Trichomonas vaginalis is an early divergent protozoan parasite that causes trichomoniasis, the most common non-viral sexually transmitted infection. In metazoans, there is abundant and detailed research on the cell cycle and the components involved in the regulation mechanisms. Regulators such as the cyclin-dependent kinases (CDKs) and cyclins acti...
Importin α, a transport factor in the classical pathway of nuclear transport of proteins in eukaryotes, has not been experimentally studied in trypanosomatids. A chimeric fluorescent version of this protein (TcImportin α-EGFP) expressed in transfected epimastigotes of Trypanosoma cruzi is characterized here. Initially, the cellular localization of...
Transcription is the first step of gene expression regulation and is a fundamental mechanism for establishing the viability and development of a cell. The TATA box-binding protein (TBP) interaction with a TATA box in a promoter is one of the best studied mechanisms in transcription initiation. TBP is a transcription factor that is highly conserved...
Trypanosomatids are a monophyletic group of parasitic flagellated protists belonging to the order Kinetoplastida. Their cytoskeleton is primarily made up of microtubules in which no actin microfilaments have been detected. Although all these parasites contain actin, it is widely thought that their actin cytoskeleton is reduced when compared to most...
The protozoan parasite Trichomonas vaginalis is a common human pathogen from one of the earliest-diverging eukaryotic lineages. At the transcriptional level, the highly conserved Inr element of RNA pol II-transcribed genes surrounds the transcription start site and is recognised by IBP39, a protein exclusive of T. vaginalis. Typical TATA boxes have...
The genome of Trypanosoma cruzi encodes for an expanded number of actins, myosins and actin binding proteins compared to Trypanosoma brucei or Leishmania spp. In T. cruzi only the expression of actin 1 (i.e. conventional actin) and profilin, an actin binding protein, has been described. In this work, the expression of a kinetoplastid-specific actin...
Giardia lamblia is one of the most common protozoan infectious agents in the world and is responsible for diarrheal disease and chronic postinfectious illness. During the host-parasite interaction, proteases are important molecules related to virulence, invasion, and colonization, not only for Giardia but also for other parasites. We aimed to chara...
The nuclear import of proteins in eukaryotic cells is a fundamental biological process. While it has been analysed to different extents in model eukaryotic organisms, this event has rarely been studied in the early divergent protozoa of the order Kinetoplastida. The work presented here represents an overview of nuclear import in these important spe...
Trypanosoma cruzi is the aetiologic agent of Chagas disease. Our research group studies ribosomal RNA (rRNA) gene transcription and nucleolus dynamics in this species of trypanosomes. RPA31 is an essential subunit of RNA polymerase I (Pol I) whose presence is apparently restricted to trypanosomes. Using fluorescent-tagged versions of this protein (...
Trypanosoma cruzi is the etiological agent of Chagas disease. Epimastigote forms of T. cruzi can be readily cultured in axenic conditions. Ethanol and dimethyl sulfoxide (DMSO) are commonly used solvents employed as vehicles for hydrophobic compounds. In order to produce a reference plot of solvent dependent growth inhibition for T. cruzi research,...
The role and regulation of actin in Trypanosoma cruzi and other related parasites is largely unknown. Based on early genome analysis, it was proposed that there was a reduced dependency on the acto-myosin system in the trypanosomatid parasites. However, more recent studies have extended the set of potential actin regulatory proteins, particularly f...
Nucleolar assembly is a cellular event that requires the synthesis and processing of ribosomal RNA, in addition to the participation of pre-nucleolar bodies (PNBs) at the end of mitosis. In mammals and plants, nucleolar biogenesis has been described in detail, but in unicellular eukaryotes it is a poorly understood process. In this study, we used l...
Trypanosoma cruzi
is the aetiological agent of Chagas disease. Our group has focused on the study of ribosomal RNA and nucleolar structure in this organism. In this work, we address the cellular location of fibrillarin in epimastigotes. As a conserved and unreported feature in trypanosomatids, fibrillarin in
T. cruzi
is encoded by two genes that di...
Leishmania major, Trypanosoma cruzi and Trypanosoma brucei are pathogenic species from the order Kinetoplastida. The molecular and cellular studies of parasites, such as of the biosynthesis of essential macromolecules, are important in designing successful strategies for control. A major stage in ribosome biogenesis is the transcription of genes en...
Chagas disease is a chronic infection that kills approximately 12,000 people a year. Mass migration of chronically infected and asymptomatic persons has caused globalization of Chagas disease and has made nonvectorial infection, including vertical and blood-borne transmission, more of a threat to human communities than vectorial infection. To contr...
Trichomonas vaginalis is a parasitic protozoan of both medical and biological relevance. Transcriptional studies in this organism have focused mainly on type II pol promoters, whereas the elements necessary for transcription by polI or polIII have not been investigated. Here, with the aid of a transient transcription system, we characterised the rD...
Trypanosoma cruzi is a species of parasitic protozoa that causes American trypanosomiasis or Chagas disease. These parasites go through a complex life cycle in Triatominae insects and vertebrate hosts. Epimastigotes are replicative forms that colonize the digestive tract of the vector and can be cultured in axenic media. The growth curve of epimast...
Synthesis of functional mRNA in eukaryotes involves processing of precursor transcripts, including the addition of a poly(A)
tail at the 3′ end. A multiprotein complex recognizes a polyadenylation signal, generally the hexanucleotide AAUAAA in metazoans,
to direct processing of the pre-mRNA. Based on sequence analysis of several cDNAs, we have prev...
The expression and biological role of actin during the Trypanosoma cruzi life cycle remains largely unknown. Polyclonal antibodies against a recombinant T. cruzi actin protein were used to confirm its expression in epimastigotes, trypomastigotes, and amastigotes. Although the overall levels of expression were similar, clear differences in the subce...
SUMMARY: Trichomonas vaginalis is a protozoan parasite causing trichomonosis, a sexually transmitted infection in humans. This parasite has numerous proteases, most of which are cysteine proteases that appear to be involved in adherence and cytotoxicity of host cells. In this report we identify and characterize a putative subtilisin-like serine pro...
Our group is interested in rRNA and ribosome biogenesis in the parasitic protozoan Trypanosoma cruzi. Epimastigotes represent an extracellular replicative stage of T. cruzi and can be cultured in axenic media. The growth curve of epimastigotes allows assessment of potential differences in the nucleoli of cells undergoing growth-rate transitions. To...
The study of genomic organization and regulatory elements of rRNA genes in metazoan paradigmatic organisms has led to the most accepted model of rRNA gene organization in eukaryotes. Nevertheless, the rRNA genes of microbial eukaryotes have also been studied in considerable detail and their atypical structures have been considered as exceptions. Ho...
The 5S ribosomal RNA (5S rRNA) is an essential component of ribosomes. Throughout evolution, variation is found among 5S rRNA genes regarding their chromosomal localization, copy number, and intergenic regions. In this report, we describe and compare the gene sequences, motifs, genomic copy number, and chromosomal localization of the Trichomonas va...
Two allelic genomic fragments containing ribosomal protein S4 encoding genes (rpS4) from Trypanosoma cruzi (CL-Brener strain) were isolated and characterized. One allele comprises two complete tandem repeats of a sequence encoding an rpS4 gene. In the other, only one rpS4 gene is found. Sequence comparison to the accessed data in the genome project...
The Trypanosoma cruzi rRNA gene promoter was characterized by deletion and point mutation analyses. A core of 89 bp was identified as the minimal region with full promoter activity. This core region is flanked upstream by a control element that stimulates its activity, and downstream by a novel down regulating region of about 200 bp. A point mutati...
The nucleolus is the main site for synthesis and processing of ribosomal RNA in eukaryotes. In mammals, plants, and yeast the nucleolus has been extensively characterized by electron microscopy, but in the majority of the unicellular eukaryotes no such studies have been performed. Here we used ultrastructural cytochemical and immunocytochemical tec...
The relative steady state concentration of mRNAs of four housekeeping single-copy type Trypanosoma cruzi genes (actin, triosephosphate isomerase, trypanothion reductase and the ribosomal protein S4) was analyzed throughout the growth curve. A distinguishable pattern was observed with maximal levels occurring at the logarithmic phase of growth and m...
Two clearly separated transcription start points (tsp) have been reported within the Trypanosoma cruzi rDNA (DNA encoding rRNA) gene spacer region. These sites are separated by 270 bp, a distance compatible with the occurrence of two core promoters. To characterize the individual participation of these two elements, a deletion analysis was carried...
Two allelic genomic fragments containing actin genes from Trypanosoma cruzi were isolated and characterized. One allele comprises two complete tandem repeats of a sequence encoding an actin gene. In the other, only one actin gene is found. Each one of these three gene copies encode for a complete and identical potential protein of 376 amino acids w...
3' Untranslated region processing and polyadenylation in Trichomonas vaginalis was analyzed by 3' rapid amplification of cDNA ends and sequence analysis of T. vaginalis mRNAs. A putative polyadenylation signal with the sequence UAAA was found 11-30 nucleotides upstream from the cleavage site. The motif pyrimidine( downward arrow)(A)(0-3)AAUU is pro...
To further study genetic heterogeneity of Mexican stocks of Trypanosoma cruzi, genomic Southern analyses from 54 Mexican isolates and 5 South American reference stocks were carried out. The membranes were hybridized with a homologous cDNA clone from the ribosomal protein S4 that identifies allelic bands from a single gene type locus. These allelic...
To further study genetic heterogeneity of Mexican stocks of Trypanosoma cruzi, genomic Southern analyses from 54 Mexican isolates and 5 South American reference stocks were carried out. The membranes were hybridized with a homologous cDNA clone from the ribosomal protein S4 that identifies allelic bands from a single gene type locus. These allelic...
The sequence of a cloned genomic fragment of Trichomonas vaginalis containing a complete actin gene was determined. An uninterrupted open reading frame of 1128 nucleotides was found that codes for an actin gene. Two overlapped consensus promoter sequences for T. vaginalis were found 12 nucleotides upstream the actin initiation codon. In addition to...
The sequence of a cloned genomic fragment of Trichomonas vaginalis containing a complete actin gene was determined. An uninterrupted open reading frame of 1128 nucleotides was found that codes for an actin gene. Two overlapped consensus promoter sequences for T. vaginalis were found 12 nucleotides upstream the actin initiation codon. In addition to...
Five Trypanosoma cruzi stocks were isolated from infected patients in the central state of Jalisco, Mexico. Parasites were isolated by direct inoculation of infected blood into BALB/c mice. The five stocks of T. cruzi were analyzed for in vitro growth, and for virulence and parasitic load in vivo. Furthermore, a genetic analysis based on restrictio...
The present work reports the in vitro biological characterization of 17 Trypanosoma cruzi isolates from southern and central México, and compares these results to those of four South American strains and one clone from Brazil. The parameters evaluated were growth rates, percentage of parasites undergoing transformation from epimastigotes to trypoma...
Two cDNA clones encoding a protein homologous to ribosomal protein S4 from the protozoan parasite Trypanosoma cruzi were isolated and characterized. Both clones potentially encode for an identical basic protein of 273 amino acids. Sequence comparisons with other species indicate that this protein is highly conserved. Hybridization studies are consi...
Two cDNA clones encoding a protein homologous to ribosomal protein S4 from the protozoan parasite Trypanosoma cruzi were isolated and characterized. Both clones potentially encode for an identical basic protein of 273 amino acids. Sequence comparisons with other species indicate that this protein is highly conserved. Hybridization studies are consi...
To improve the selection phenotype of the expression plasmid pTEX, a Trypanosoma cruzi rDNA (DNA coding for rRNA) gene spacer fragment (806 bp) containing a mapped transcription start point (tsp) was cloned in the vectors pTEX and pTEX-cat, generating the plasmids pRIBOTEX and pRIBOTEX-cat. T. cruzi cultures transiently transfected with pRIBOTEX-ca...
To determine the occurrence of conserved domains of presumed functional selection, a genomic restriction analysis was carried out in the region surrounding a transcription start point (tsp) from the rRNA cistron in T. cruzi. The transcribed spacer was found highly conserved among several isolates, whereas at 146 bp upstream from the tsp a highly po...
The nucleotide (nt) sequence (2106 bp) of a cloned rDNA (encoding ribosomal RNA) spacer region from Trypanosoma cruzi was determined and a putative transcription start point (tsp) was mapped. The assigned length for the transcribed spacer is 1768 bp and its tsp is present 270-bp upstream from an alternative tsp published for the equivalent gene fro...
Our research work group has been interested in the study of the ribosomal RNA and 5S gene systems from Trypanosoma cruzi. Our contributions span from the general description of a multifragmented molecular system, to the sequence analysis of some ribosomal RNA coding regions. From the latter, we have constructed phylogenetic trees of the Trypanosoma...
Seventeen Mexican Trypanosoma cruzi stocks and five South American reference strains were analyzed by Hind III restriction fragment length polymorphisms associated with ribosomal RNA gene spacers and by HinfI digestion patterns of total DNA. Our findings demonstrate the occurrence of genetic heterogeneity within these stocks. Hierarchic and non-hie...
To further study the ribosomal RNA genetic system in Trypanosoma cruzi, the 5S rRNA gene family was characterized. We found that this gene family is reiterated about 1600 times per diploid nuclei and is mostly organized as a tandem repeat of 481 base pairs. These gene clusters were assigned to two chromosomes of about 1500 and 1400 kilobase pairs....
Abstract To further study the ribosomal RNA genetic system in Trypanosoma cruzi, the 5S rRNA gene family was characterized. We found that this gene family is reiterated about 1600 times per diploid nuclei and is mostly organized as a tandem repeat of 481 base pairs. These gene clusters were assigned to two chromosomes of about 1500 and 1400 kilobas...
A Trypanosoma cruzi small subunit ribosomal RNA gene was sequenced from genomic recombinant plasmid clones. The assigned coding region was 2319 bp, the longest SSU rRNA gene described to date. On the basis of comparisons with published sequences from Crithidia fasciculata, Trypanosoma brucei, and Leishmania donovani, we conclude that the extra nucl...
Genomic and cDNA actin clones were isolated from Taenia solium gene libraries. The actin genes are interrupted by intervening sequences. Protein coding regions of both genes predict the same amino acid sequence. cDNA sequence data indicate that at least one gene is expressed at the larval stage. Results from Northern and Western blots showed that T...
To further analyze the organization of the nuclear rDNA locus in Trypanosoma cruzi, genomic recombinant plasmid clones were constructed and isolated after hybridization with rRNA molecules as hybridization probes. Approximately 11 kilobase pairs from the cistron were cloned in three recombinant plasmids carrying adjacent genomic fragments. Restrict...
Trypanosoma cruzi ribosomal RNA was analyzed electrophoresis. On agarose gels, where both large- and small-size species are grossly fractionable, it revealed two bands in the small-size region. These were similar in size to the mammalian 5.8 S and 5 S species. Increased resolution, however, showed these two bands to be composite. The pseudo 5.8 S b...
The location of ribosomal RNA genes in total-nuclear-enriched DNA preparations of Trypanosoma cruzi was analyzed by using restriction endonucleases and the eight cytoplasmic ribosomal RNA species of this organism. Two contiguous SstI DNA fragments of about 9.88 and 1.7 kilobase pairs contained the three large-size ribosomal RNA species, 18 S, beta,...
The large-molecular-mass ribosomal ribonucleic acid from Trypanosoma cruzi probably contains an internal break. The molecule can be obtained in its intact form or in its two fragments depending on the denaturing agents used for its purification and/or display. This break appears to be an in vivo late processing step rather than a random nucleolytic...
