Cloning and characterization of the gene encoding Trypanosoma cruzi DNA topoisomerase II (original) (raw)
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Molecular and Biochemical Parasitology, 2003
The protozoan parasite Trypanosoma cruzi is the causal agent of American trypanosomiasis or Chagas disease, which afflicts millions of people in Central and South America. Its life cycle involves at least three distinct developmental stages: epimastigotes, trypomastigotes and amastigotes . The epimastigote forms replicate in the midgut of the insect host and develop into non-replicative metacyclic trypomastigote forms by the process of metacyclogenesis. Metacyclic trypomastigotes are released in the excreta of insects of the Reduviidae family (triatomine insects) during feeding and invade the cells of the mammalian host. Within the host cells, the parasite differentiates into the replicative amastigote form, which in turn differentiates into bloodstream trypomastigotes, which infect new cells.
Gene organization and sequence analyses of transfer RNA genes in Trypanosomatid parasites
BMC Genomics, 2009
The protozoan pathogens Leishmania major, Trypanosoma brucei and Trypanosoma cruzi (the Tritryps) are parasites that produce devastating human diseases. These organisms show very unusual mechanisms of gene expression, such as polycistronic transcription. We are interested in the study of tRNA genes, which are transcribed by RNA polymerase III (Pol III). To analyze the sequences and genomic organization of tRNA genes and other Pol III-transcribed genes, we have performed an in silico analysis of the Tritryps genome sequences.
Journal of Biological Chemistry, 1998
In previous works we have identified genes in the protozoan parasite Trypanosoma cruzi whose structure resemble those of mammalian mucin genes. Indirect evidence suggested that these genes might encode the core protein of parasite mucins, glycoproteins that were proposed to be involved in the interaction with, and invasion of, mammalian host cells. We now show that the mucin gene family from T. cruzi is much larger and diverse than expected. A minimal number of 484 mucin genes per haploid genome is calculated for a parasite clone. Most, if not all, genes are transcribed, as deduced from cDNA analysis. Comparison of the cDNA sequences showed evidences of a high mutation rate in localized regions of the genes. Sequence conservation among members of the family is much higher in the untranslated (UTR) regions than in the sequences encoding the mature mucin core protein. Transcription units can be classified into two main subfamilies according to the sequence homologies in the 5-UTR, whereas the 3-UTR is highly conserved in all clones analyzed. The common origin of members of this gene family as well as their relationships can be defined by sequence comparison of different domains in the transcription units. The regions encoding the N and C termini, supposed to correspond to the leader peptide and membrane-anchoring signal, respectively, (Di Noia, J. M., Sá nchez, D. O., and Frasch, A. C. C. (1995) J. Biol. Chem. 270, 24146 -24149) are highly conserved. Conversely, the central regions are highly variable. These regions encode the target sites for O-glycosylation and are made of a variable number of repetitive units rich in Thr and Pro residues or are nonrepetitive but still rich in Thr/Ser and Pro residues. The region putatively coding for the N-terminal domain of the mature core protein is hypervariable, being different in most of the transcripts sequenced. Nonrepetitive central domains are unique to each gene. Gene-specific probes show that the relative abundance of different mRNAs varies greatly within the same parasite clone.
The Journal of biological chemistry, 2008
Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. The PUF (Pumilio/ FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypanosomatids. We used a ribonomic approach to identify the putative target mRNAs associated with TcPUF6, a member of the T. cruzi PUF family. TcPUF6 is expressed in discrete sites in the cytoplasm at various stages of the parasite life cycle and is not associated with the translation machinery. The overexpression of a tandem affinity purification-tagged TcPUF6 protein allowed the identification of associated mRNAs by affinity purification assays and microarray hybridization yielding nine putative target mRNAs. Whole expression analysis of transfected parasites showed that the mRNAs associated with TcPUF6 were down-regulated in populations overexpressing TcPUF6. The association of TcPUF6 with the TcDhh1 helicase in vivo and the cellular co-localization of these proteins in epimastigote forms suggest that TcPUF6 promotes degradation of its associated mRNAs through interaction with RNA degradation complexes. Analysis of the mRNA levels of the putative TcPUF6regulated genes during the parasite life cycle showed that their transcripts were up-regulated in metacyclic trypomastigotes. In these infective forms no co-localization between TcPUF6 and TcDhh1 was observed. Our results suggest that TcPUF6 regulates the half-lives of its associated transcripts via differential association with mRNA degradation complexes throughout its life cycle.
Biochemical and Biophysical Research Communications, 2012
Background: In rural areas of Espírito Santo state, southeast Brazil, triatomine species attracted by light frequently invade residences. The aim of this study was to investigate the Trypanosoma cruzi discrete typing units (DTUs) harbored by these triatomines. Methods: Triatomine's intestinal contents were examined, inoculated in mice, and the positive samples were cultivated. Flagellates obtained from infected mice hemoculture were submitted to DNA extraction using a salting-out method and to TcSC5D gene amplification. The amplified samples were sequenced, and polymorphism was analyzed for DTU identification. Results: Three hundred and ninety-four triatomines were identified: Triatoma vitticeps (90.03%), Panstrongylus geniculatus (8.89%), Panstrongylus megistus (0.54%), Panstrongylus diasi (0.27%), and Triatoma tibiamaculata (0.27%). Among the specimens, 251/394 (67.65%) presented flagellated forms similar to T. cruzi. After triatomine intestinal content inoculation into mice, 134 mice presented T. cruzi-like trypomastigotes from Tr. vitticeps and P. geniculatus and 89 samples were positive in hemoculture. Sixty-two samples were analyzed for the TcSC5D gene and TcI, TcII, TcIII, and TcIV DTUs were identified. Conclusions: We observed T. cruzi DTU diversity in Tr. vitticeps and P. geniculatus, which showed the predominance of TcII and occurrence of TcI, TcIII and TcIV. Triatomines presented high T. cruzi infection rates. Since little is known regarding the possible mammalian hosts that maintain the T. cruzi cycle, further studies are necessary to obtain a better understanding of the parasite transmission cycle in this region.
1998
We have initiated large-scale sequencing of the third smallest chromosome of the CL Brener strain of Trypanosoma cruzi and we report here the complete sequence of a contig consisting of three cosmids. This contig covers 93.4 kb and has been found to contain 20-30 novel genes and several repeat elements, including a novel chromosome 3-specific 400-bp repeat sequence. The intergenic sequences were found to be rich in di-and trinucleotide repeats of varying lengths and also contained several known T. cruzi repeat elements. The sequence contains 29 open reading frames (ORFs) longer than 700 bp, the longest being 5157 bp, and a large number of shorter ORFs. Of the long ORFs, seven show homology to known genes in parasites and other organisms, whereas four ORFs were confirmed by sequencing of cDNA clones. Two shorter ORFs were confirmed by a database homology and a cDNA clone, respectively, and one RNA gene was identified. The identified genes include two copies of the gene for alanine-aminotransferase as well as genes for glucose-6-phosphate isomerase, protein kinases and phosphatases, and an ATP synthase subunit. An interesting feature of the sequence was that the genes appear to be organized in two long clusters containing multiple genes on the same strand. The two clusters are transcribed in opposite directions and they are separated by an ∼20-kb long, relatively GC-rich sequence, that contains two large repetitive elements as well as a pseudogene for cruzipain and a gene for U2snRNA. It is likely that this strand switch region contains one or more regulatory and promoter regions. The reported sequence provides the first insight into the genome organization of T. cruzi and shows the potential of this approach for rapid identification of novel genes.
bioRxiv (Cold Spring Harbor Laboratory), 2014
The TcZFPs are a family of small zinc finger proteins harboring WW domains or Proline rich motifs. In Trypanosoma brucei, ZFPs are involved during stage specific differentiation. TcZFPs interact with each other using the WW domain (ZFP2 and ZFP3) and the proline rich motif (ZFP1). The tcZFP1b member is exclusive to Trypanosoma cruzi and it is only expressed in trypomastigote stage. We used a tetracycline inducible vector to express ectopically tcZFP1b in the epimastigote stage. Upon induction of tcZFP1b, the parasites stopped dividing completely after five days. Visual inspection showed abnormal distortedmorphology (monster) cells with multiple flagella and increased DNA contents. We were interested in investigate global transcription changes occurred during the generation of this abnormal phenotype. Thus, we performed RNA-seq transcriptome profiling with a 454 pyrosequencer to analyze the global changes after ectopic expression of tcZFP1b. The total mRNAs sequenced from induced and non-induced control epimastigotes showed, after filtering the data, a set of 70 genes having equal or more than 3X fold change upregulation, while 35 genes showed equal or more than 3X fold downregulation. Interestingly, several transsialidase-like genes and pseudogenes were upregulated along with several genes in the categories of amino acid catabolism and carbohydrate metabolism. On the other hand, hypothetical proteins, fatty acid biosynthesis and mitochondrial functions dominated the group of downregulated genes. Our data showed that several mRNAs sharing related functions and pathways changed their levels in a concerted pattern resembling post-transcriptional regulons. We also found two different motifs in the 3´UTRs of the majority of mRNAs, one for upregulated and other for downregulated genes .