| 1. |
- Davids, Barbara J., et al.
(författare)
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Identification of Conserved Candidate Vaccine Antigens in the Surface Proteome of Giardia lamblia
- 2019
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Ingår i: Infection and Immunity. - : AMER SOC MICROBIOLOGY. - 0019-9567 .- 1098-5522. ; 87:6
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Tidskriftsartikel (refereegranskat)abstract
- Giardia lamblia, one of the most common protozoal infections of the human intestine, is an important worldwide cause of diarrheal disease, malabsorption, malnutrition, delayed cognitive development in children, and protracted postinfectious syndromes. Despite its medical importance, no human vaccine is available against giardiasis. A crude veterinary vaccine has been developed, and experimental vaccines based on expression of multiple variant-specific surface proteins have been reported, but poorly defined vaccine components and excessive antigen variability are problematic for pharmaceutical vaccine production. To expand the repertoire of antigen candidates for vaccines, we reasoned that surface proteins may provide an enriched source of such antigens since key host effectors, such as secretory IgA, can directly bind to such antigens in the intestinal lumen and interfere with epithelial attachment. Here, we have applied a proteomics approach to identify 23 novel surface antigens of G. lamblia that show >90% amino acid sequence identity between the two human-pathogenic genetic assemblages (A and B) of the parasite. Surface localization of a representative subset of these proteins was confirmed by immunostaining. Four selected proteins, uridine phosphorylase-like protein-1, protein 21.1 (GL50803_ 27925), alpha 1-giardin, and alpha 11-giardin, were subsequently produced in recombinant form and shown to be immunogenic in mice and G. lamblia-infected humans and confer protection against G. lamblia infection upon intranasal immunization in rodent models of giardiasis. These results demonstrate that identification of conserved surface antigens provides a powerful approach for overcoming a key rate-limiting step in the design and construction of an effective vaccine against giardiasis.
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| 2. |
- El-Sayed, Najib M., et al.
(författare)
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The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease.
- 2005
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Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 1095-9203 .- 0036-8075. ; 309:5733, s. 409-15
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Tidskriftsartikel (refereegranskat)abstract
- Whole-genome sequencing of the protozoan pathogen Trypanosoma cruzi revealed that the diploid genome contains a predicted 22,570 proteins encoded by genes, of which 12,570 represent allelic pairs. Over 50% of the genome consists of repeated sequences, such as retrotransposons and genes for large families of surface molecules, which include trans-sialidases, mucins, gp63s, and a large novel family (>1300 copies) of mucin-associated surface protein (MASP) genes. Analyses of the T. cruzi, T. brucei, and Leishmania major (Tritryp) genomes imply differences from other eukaryotes in DNA repair and initiation of replication and reflect their unusual mitochondrial DNA. Although the Tritryp lack several classes of signaling molecules, their kinomes contain a large and diverse set of protein kinases and phosphatases; their size and diversity imply previously unknown interactions and regulatory processes, which may be targets for intervention.
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| 3. |
- Ferella, Marcela, et al.
(författare)
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A solanesyl-diphosphate synthase localizes in glycosomes of Trypanosoma cruzi
- 2006
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 281:51, s. 39339-39348
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Tidskriftsartikel (refereegranskat)abstract
- We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence ( molecular mass similar to 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target.
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| 4. |
- Ferella, Marcela
(författare)
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Detection and characterization of novel proteins in Trypanosoma cruzi
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Trypanosoma cruzi is a flagellated protozoan parasite. It infects a wide range of mammals, including humans. Human T. cruzi infections are endemic to South and Central America. The parasite is transmitted to humans mainly through an insect from the Triatominae subfamily, which feeds from mammals and defecates at the site of the wound, which allows the parasites in the faeces to infect the damaged cells at the bite area. The disease is named Chagas disease in honour of Carlos Chagas who first detected the parasites in the insect, and in this way determined that the vector of the disease was the triatomine bug. There is no cure for Chagas disease and if it is left untreated it can be lethal in the initial stage of the infection, especially for children. If the affected patients develop the chronic form of the disease, there is a high risk of organ deterioration due to the long term presence of the parasites. Only two drugs are used at present to treat Chagas disease, Nifurtimox and Benznidazole. These drugs were developed in 1960s-70s and they can cause severe side effects. Although preventive and control measures have been effective to reduce transmission, there are still 15,000 deaths per year and over 20 million people are at risk of contracting the disease. Chagas disease is one of the socalled neglected tropical diseases, for which there is little interest from pharmaceutical companies to develop new drugs. In response to the critical need for new safe and effective drugs, much research has been performed by many groups in the field, in order to expand the knowledge on T. cruzi biology and to study different enzymes and metabolic pathways that differ from humans. My aim in this thesis was to detect novel proteins in T. cruzi and characterize them by means of: assessing their localization, determining their enzymatic activity, infering putative identity, if unknown by homology comparisons, and determine if they were really expressed in the parasite. In paper I, we detected a polyprenyl synthase in the T. cruzi EST database. We have expressed and characterized this protein. The enzymes of the polyprenyl synthase family are involved in the synthesis of isoprenoids, which are essential for cell function. The identified protein was a solanesyl diphosphate synthase (TcSPPS) that had all the conserved motifs of the family, presented polyprenyl synthase activity and synthesized the maximum chain isoprenoid, solanesyl diphosphate. Long chain isoprenoids are used in ubiquinone biosynthesis. This was shown by the ability of TcSPPS to complement an E. coli strain deficient for ubiquinone production. By using immunofluorescence microscopy, immunogold electron microscopy and cell fractionation we localized TcSPPS to the glycosomes, a peroxisome-like organelle of T. cruzi. In paper II, we report the localization of a short polyprenyl synthase, farnesyl diphosphate synthase (FPPS) in both T. cruzi and Trypanosoma brucei, a closely related trypanosome to T. cruzi that causes sleeping sickness in Africa. Short chain polyprenyl synthases produce short isoprenoids that are utilized to for example modify signalling proteins that utilize the isoprenoid arm to attach to membranes and receptors. As we found the TcSPPS in the glycosomes, we wanted to determine if the entire part of the isoprenoid pathway where these two enzymes take part, was compartmentalized to the glycosomes or not. We found that this was not the case. Both TcFPPS and TbFPPS are present in the cytoplasm. In paper III, we partially characterized a third polyprenyl synthase of T. cruzi, TcPPS. We detected this protein by western blots analysis in the three stages of the parasite and in the cytoplasm of epimastigotes in T. cruzi. It is an unusual protein, due to the 747 amino acid sequence and a molecular weight of 85 kDa, compared to the usual size for the family, which is around 40 kDa. Another particular feature was the presence of a domain of unknown function, DUF2006, which is unrelated to the conserved polyprenyl synthase conserved motifs. We hypothesized that this enzyme could be a GGPPS. The recombinant TcPPS had polyprenyl activity, but the preferred substrate was GPP instead of FPP, the preferred substrates of GGPPSs. In paper IV, we used a mass spectrometry based proteomic approach to detect novel proteins in an organelle enriched sample from T. cruzi epimastigotes. The organellar proteins were separated by 2DGE and 1DGE and subsequently subjected to LC-MS/MS. The results from mass spectrometry were used to search against the T. cruzi translated genome. The search rendered 396 protein identifications. For 173 of them, this was the first expression data reported in T. cruzi. Furthermore, the proteins in the sample belonged to several organellar compartments and the level of cytoplasmic and highly abundant surface proteins was much reduced. We located five novel proteins to the acidocalcisome, mitochondrion, ER and cytoplasmic vesicles, through immunofluorescence microscopy of epitopetagged over-expressed clones. In summary, this work has contributed to the detection and characterization of several novel proteins in T. cruzi, and has answered various questions and has generated new hypotheses to be tested.
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| 5. |
- Ferella, Marcela, et al.
(författare)
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Gene expression changes during Giardia-host cell interactions in serum-free medium
- 2014
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Ingår i: Molecular and biochemical parasitology (Print). - : Elsevier BV. - 0166-6851 .- 1872-9428. ; 197:1-2, s. 21-23
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Tidskriftsartikel (refereegranskat)abstract
- Serial Analysis of Gene Expression (SAGE) was used to quantify transcriptional changes in Giardia intestinalis during its interaction with human intestinal epithelial cells (IECs, HT-29) in serum free M199 medium. Transcriptional changes were compared to those in trophozoites alone in M199 and in TYI-S-33 Giardia growth medium. In total, 90 genes were differentially expressed, mainly those involved in cellular redox homeostasis, metabolism and small molecule transport but also cysteine proteases and structural proteins of the giardin family. Only 29 genes changed their expression due to IEC interaction and the rest were due to M199 medium. Although our findings generated a small dataset, it was consistent with our earlier microarray studies performed under different interaction conditions. This study has confined the number of genes in Giardia to a small subset that specifically change their expression due to interaction with IECs.
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| 6. |
- Franzen, Oscar, et al.
(författare)
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The Short Non-Coding Transcriptome of the Protozoan Parasite Trypanosoma cruzi
- 2011
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Ingår i: PLoS Neglected Tropical Diseases. - : Public Library of Science (PLoS). - 1935-2727 .- 1935-2735. ; 5:8, s. e1283-
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Tidskriftsartikel (refereegranskat)abstract
- The pathway for RNA interference is widespread in metazoans and participates in numerous cellular tasks, from gene silencing to chromatin remodeling and protection against retrotransposition. The unicellular eukaryote Trypanosoma cruzi is missing the canonical RNAi pathway and is unable to induce RNAi-related processes. To further understand alternative RNA pathways operating in this organism, we have performed deep sequencing and genome-wide analyses of a size-fractioned cDNA library (16-61 nt) from the epimastigote life stage. Deep sequencing generated 582,243 short sequences of which 91% could be aligned with the genome sequence. About 95-98% of the aligned data (depending on the haplotype) corresponded to small RNAs derived from tRNAs, rRNAs, snRNAs and snoRNAs. The largest class consisted of tRNA-derived small RNAs which primarily originated from the 3' end of tRNAs, followed by small RNAs derived from rRNA. The remaining sequences revealed the presence of 92 novel transcribed loci, of which 79 did not show homology to known RNA classes.
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| 7. |
- Franzén, Oscar, et al.
(författare)
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Transcriptome Profiling of Giardia intestinalis Using Strand-specific RNAseq
- 2013
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Ingår i: PloS Computational Biology. - : Public Library of Science (PLoS). - 1553-734X .- 1553-7358. ; 9:3
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Tidskriftsartikel (refereegranskat)abstract
- Giardia intestinalis is a common cause of diarrheal disease and it consists of eight genetically distinct genotypes or assemblages (A-H). Only assemblages A and B infect humans and are suggested to represent two different Giardia species. Correlations exist between assemblage type and host-specificity and to some extent symptoms. Phenotypical differences have been documented between assemblages and genome sequences are available for A, B and E. We have characterized and compared the polyadenylated transcriptomes of assemblages A, B and E. Four genetically different isolates were studied (WB (AI), AS175 (AII), P15 (E) and GS (B)) using paired-end, strand-specific RNA-seq. Most ofthe genome was transcribed in trophozoites grown in vitro, but at vastly different levels.RNA-seq confirmed many of the present annotations and refined the current genome annotation. Gene expression divergence was found to recapitulate the known phylogeny, and uncovered lineage-specific differences in expression. Polyadenylation sites were mapped for over 70% of the genes and revealed many examples of conserved and unexpectedly long 3' UTRs. 28 open reading frames were found in a non-transcribed gene cluster on chromosome 5 of the WB isolate. Analysis of allele-specific expression revealed a correlation between allele-dosage and allele expression in the GS isolate. Previously reported cis-splicing events were confirmed and global mapping of cis-splicing identified only one novel intron. These observations can possibly explain differences in host-preference and symptoms, and it will be the basis for further studies of Giardia pathogenesis and biology.
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| 8. |
- Lai, De-Hua, et al.
(författare)
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Solanesyl Diphosphate Synthase, an Enzyme of the Ubiquinone Synthetic Pathway, Is Required throughout the Life Cycle of Trypanosoma brucei
- 2014
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Ingår i: Eukaryotic Cell. - 1535-9778 .- 1535-9786. ; 13:2, s. 320-328
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Tidskriftsartikel (refereegranskat)abstract
- Ubiquinone 9 (UQ9), the expected product of the long-chain solanesyl diphosphate synthase of Trypanosoma brucei (TbSPPS), has a central role in reoxidation of reducing equivalents in the mitochondrion of T. brucei. The ablation of TbSPPS gene expression by RNA interference increased the generation of reactive oxygen species and reduced cell growth and oxygen consumption. The addition of glycerol to the culture medium exacerbated the phenotype by blocking its endogenous generation and excretion. The participation of TbSPPS in UQ synthesis was further confirmed by growth rescue using UQ with 10 isoprenyl subunits (UQ10). Furthermore, the survival of infected mice was prolonged upon the downregulation of TbSPPS and/or the addition of glycerol to drinking water. TbSPPS is inhibited by 1-[(n-oct-1-ylamino)ethyl] 1,1-bisphosphonic acid, and treatment with this compound was lethal for the cells. The findings that both UQ9 and ATP pools were severely depleted by the drug and that exogenous UQ10 was able to fully rescue growth of the inhibited parasites strongly suggest that TbSPPS and UQ synthesis are the main targets of the drug. These two strategies highlight the importance of TbSPPS for T. brucei, justifying further efforts to validate it as a new drug target.
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| 9. |
- Peirasmaki, Dimitra, et al.
(författare)
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High Cysteine Membrane Proteins (HCMPs) Are Up-Regulated DuringGiardia-Host Cell Interactions
- 2020
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Ingår i: Frontiers in Genetics. - : FRONTIERS MEDIA SA. - 1664-8021. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Giardia intestinaliscolonizes the upper small intestine of humans and animals, causing the diarrheal disease giardiasis. This unicellular eukaryotic parasite is not invasive but it attaches to the surface of small intestinal epithelial cells (IECs), disrupting the epithelial barrier. Here, we used anin vitromodel of the parasite's interaction with host IECs (differentiated Caco-2 cells) and RNA sequencing (RNAseq) to identify differentially expressed genes (DEGs) inGiardia, which might relate to the establishment of infection and disease induction.Giardiatrophozoites interacted with differentiated Caco-2 cells for 1.5, 3, and 4.5 h and at each time point, 61, 89, and 148 parasite genes were up-regulated more than twofold, whereas 209, 265, and 313 parasite genes were down-regulated more than twofold. The most abundant DEGs encode hypothetical proteins and members of the High Cysteine Membrane Protein (HCMP) family. Among the up-regulated genes we also observed proteins associated with proteolysis, cellular redox balance, as well as lipid and nucleic acid metabolic pathways. In contrast, genes encoding kinases, regulators of the cell cycle and arginine metabolism and cytoskeletal proteins were down-regulated. Immunofluorescence imaging of selected, up-regulated HCMPs, using C-terminal HA-tagging, showed localization to the plasma membrane and peripheral vesicles (PVs). The expression of the HCMPs was affected by histone acetylation and free iron-levels. In fact, the latter was shown to regulate the expression of many putative giardial virulence factors in subsequent RNAseq experiments. We suggest that the plasma membrane localized and differentially expressed HCMPs play important roles duringGiardia-host cell interactions.
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| 10. |
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| 11. |
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| 12. |
- Respuela, Patricia, 1977-, et al.
(författare)
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Distinct histone modification profiles at transcription start sites during development of the parasite Trypanosoma cruzi.
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The parasite Trypanosoma cruzi is the etiological agent causing Chagas' disease in Central and South America. This protozoon presents a complex life cycle that comprises three major developmental stages as the parasite is transmitted from the invertebrate vector to a mammalian host. Such drastic environmental changes demand rapid adjustment of the parasite cellular processes. This adaptation appears to be mainly achieved by regulating gene expression levels through post-transcriptional processes. We have previously shown the important role of chromatin in regulating transcriptional initiation through histone modifications. Histones typically associated with active chromatin states (i.e. histone acetylation and histone H3 trimethylated K4) were preferentially enriched at sequences directly flanking transcription start sites in the intergenic region between divergently transcribed polycistronic gene clusters. In this study, we compare acetylated histone H3 profiles in replicative (i.e. logarithmic epimastigotes) and non-replicative (i.e. stationary epimastigotes and trypomastigotes) parasite stages. Histone acetylation was found less abundant in non-replicative stages consistent with previous observations of diminished transcriptional activity in these stages and suggesting potential links between transcription and replication. This was also accompanied by possible changes in nucleosome positioning. We also report the epigenetic profile of histone H3 trimethylated K36, a histone modification previously uncharacterized in T. cruzi . This histone mark was found enriched at regions of transcriptional initiation, suggesting a rather distinct function compared with its established roles in other organisms.
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| 13. |
- Respuela, Patricia, 1977-, et al.
(författare)
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DNA methylation patterns in the parasite Trypanosoma cruzi
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Although DNA methylation is found to be a major epigenetic regulator of vital cellular processes in most organisms, its role in the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas' disease, remains largely unknown. In this study, we report detection of 5-methyl cytosine (m5C) in T. cruzi DNA and also determine the genomic distribution of m5C by methyl DNA immunoprecipitation (MeDIP). Some of the methylated genomic loci identified were within repetitive retrotransposons or pseudogenes, suggesting a probable role for DNA methylation in transcriptional silencing similar to what has been reported in various organisms. DNA methylation was also found at intergenic regions close to putative transcription start sites, as well as in regions of transcriptional termination, suggesting that these loci are characterized by a complex combination of activating (e.g. acH3, H3K4me3) and silencing (e.g. m5C) epigenetic modifications. Interestingly, both immunostaining and immunoprecipitation results clearly demonstrated that DNA methylation is especially abundant in kinetoplast minicircles DNA. Both global and locus-specific differences in DNA methylation were observed between non-replicative (i.e. trypomastigotes, stationary-phase epimastigotes) and replicative (i.e. logarithmic epimastigotes) stages of the parasite, suggesting a role for DNA methylation in T. cruzi development.
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| 14. |
- Respuela, Patricia, 1977-, et al.
(författare)
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Histone acetylation and methylation at sites initiating divergent polycistronic transcription in Trypanosoma cruzi
- 2008
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 283:23, s. 15884-15892
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Tidskriftsartikel (refereegranskat)abstract
- Trypanosomes are ancient eukaryotic parasites in which the protein-coding genes, organized in large polycistronic clusters on both strands, are transcribed from as yet unidentified promoters. In an effort to reveal transcriptional initiation sites, we examined the Trypanosoma cruzi genome for histone modification patterns shown to be linked to active genes in various organisms. Here, we show that acetylated and methylated histones were found to be enriched at strand switch regions of divergent gene arrays, not at convergent clusters or intra- and intergenic regions within clusters. The modified region showed a bimodular profile with two peaks centered over the 5'-regions of the gene pair flanking the strand switch region. This pattern, which demarcates polycistronic transcription units originating from bidirectional initiation sites, is likely to be common in kinetoplastid parasites as well as in other organisms with polycistronic transcription. In contrast, no acetylation was found at promoters of the highly expressed rRNA and spliced leader genes or satellite DNA or at tested retrotransposonal elements. These results reveal, for the first time, the presence of specific epigenetic marks in T. cruzi with potential implications for transcriptional regulation; they indicate that both histone modifications and bidirectional transcription are evolutionarily conserved.
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