Renata Souza - Academia.edu (original) (raw)

Papers by Renata Souza

Infection and Immunity, 2007

A member of the Trypanosoma cruzi gp82 family, expressed on metacyclic trypomastigote surface and... more A member of the Trypanosoma cruzi gp82 family, expressed on metacyclic trypomastigote surface and identified by monoclonal antibody (MAb) 3F6, plays a key role in host cell invasion. Apart from the gp82 defined by MAb 3F6, no information is available on members of this protein family. From cDNA clones encoding gp82 proteins sharing 59.1% sequence identity, we produced the recombinant

PLoS ONE, 2011

Background: Trypanosoma cruzi has a single flagellum attached to the cell body by a network of sp... more Background: Trypanosoma cruzi has a single flagellum attached to the cell body by a network of specialized cytoskeletal and membranous connections called the flagellum attachment zone. Previously, we isolated a DNA fragment (clone H49) which encodes tandemly arranged repeats of 68 amino acids associated with a high molecular weight cytoskeletal protein.

International Journal for Parasitology, 2014

The identification of new targets for vaccine and drug development for the treatment of Chagas&am... more The identification of new targets for vaccine and drug development for the treatment of Chagas' disease is dependent on deepening our understanding of the parasite genome. Vectors for genetic manipulation in Trypanosoma cruzi basically include those that remain as circular episomes and those that integrate into the parasite's genome. Artificial chromosomes are alternative vectors to overcome problematic transgene expression often occurring with conventional vectors in this parasite. We have constructed a series of vectors named pTACs (Trypanosome Artificial Chromosomes), all of them carrying telomeric and subtelomeric sequences and genes conferring resistance to different selection drugs. In addition, one pTAC harbours a modified GFP gene (pTAC-gfp), and another one carries the ornithine decarboxilase gene from Crithidia fasciculata (pTAC-odc). We have encountered artificial chromosomes generated from pTACs in transformed T. cruzi epimastigotes for every version of the designed vectors. These extragenomic elements, in approximately 6-8 copies per cell, remained as linear episomes, contained telomeres and persisted after 150 and 60 generations with or without selection drugs, respectively. The linear molecules remained stable through the different T. cruzi developmental forms. Furthermore, derived artificial chromosomes from pTAC-odc could complement the auxotrophy of T. cruzi for polyamines. Our results show that pTACs constitute useful tools for reverse functional genetics in T. cruzi that will contribute to a better understanding of T. cruzi biology.

Infection and Immunity, 2007

A member of the Trypanosoma cruzi gp82 family, expressed on metacyclic trypomastigote surface and... more A member of the Trypanosoma cruzi gp82 family, expressed on metacyclic trypomastigote surface and identified by monoclonal antibody (MAb) 3F6, plays a key role in host cell invasion. Apart from the gp82 defined by MAb 3F6, no information is available on members of this protein family. From cDNA clones encoding gp82 proteins sharing 59.1% sequence identity, we produced the recombinant proteins J18 and C03, the former containing and the latter lacking the epitope for MAb 3F6. Polyclonal antibodies to J18 and C03 proteins were generated and used, along with MAb 3F6, to analyze the expression and cellular localization of gp82 family members in metacyclic forms of CL and G strains, which belong to highly divergent genetic groups. By two-dimensional gel electrophoresis and immunoblotting, molecules of 82 to 86 kDa, focusing at pH 4.6 to 5.4, and molecules of 72 to 88 kDa, focusing at pH 4.9 to 5.7, were visualized in CL and G strains, respectively. Flow cytometry and microscopic analysis revealed in both strains similar expression of MAb 3F6-reactive gp82 in live and permeabilized parasites, indicating its surface localization. The reaction of live parasites of both strains with anti-J18 antibodies was weaker than with MAb 3F6 and was increased by permeabilization. Anti-C03 antibodies bound predominantly to flagellar components in permeabilized G strain parasites, but in the CL strain the flagellum was not the preferential target for these antibodies. Host cell invasion of metacyclic forms was inhibited by J18 protein, as well as by MAb 3F6 and anti-J18 antibodies, but not by C03 protein or anti-C03 antibodies.

Gene, 2003

We present a useful refinement of the molecular karyotype of clone CL Brener, the reference clone... more We present a useful refinement of the molecular karyotype of clone CL Brener, the reference clone of the Trypanosoma cruzi Genome Project. The assignment of 210 genetic markers (142 expressed sequence tags (ESTs), seven cDNAs, 32 protein-coding genes, eight sequence tagged sites (STSs), 21 repetitive sequences) to the chromosomal bands separated by pulsed field gel electrophoresis (PFGE) identified 61 chromosome-specific markers, two size-polymorphic chromosomes and seven linkage groups. Fourteen new repetitive elements were isolated in this work and mapped to the chromosomal bands. We found that at least ten repetitive elements can be mapped to each chromosomal band, which may render the whole genome sequence assembly a difficult task. To construct the integrated map of chromosomal band XX, we used yeast artificial chromosome (YAC) overlapping clones and a variety of probes (i.e. known gene sequences, ESTs, STSs generated from the YAC ends). The total length covered by the YAC contig was approximately 1.3 Mb, covering 37% of the entire chromosome. We found some degree of polymorphism among YACs derived from band XX. These results are in agreement with data from phylogenetic analysis of T. cruzi which suggest that clone CL Brener is a hybrid genotype [Mol. Biochem. Parasitol. 92 (1998) 253; Proc. Natl. Acad. Sci. USA 98 ]. The physical map of the chromosomal bands, together with the isolation of specific chromosomal markers, will contribute in the global effort to sequence the nuclear genome of this parasite. q

Eukaryotic Cell, 2007

A new family of site-specific repeated elements identified in Trypanosoma cruzi, which we named T... more A new family of site-specific repeated elements identified in Trypanosoma cruzi, which we named TcTREZO, is described here. TcTREZO appears to be a composite repeated element, since three subregions may be defined within it on the basis of sequence similarities with other T. cruzi sequences. Analysis of the distribution of TcTREZO in the genome clearly indicates that it displays site specificity for insertion. Most TcTREZO elements are flanked by conserved sequences. There is a highly conserved 68-bp sequence at the 5 end of the element and a sequence domain of ϳ500 bp without a well-defined borderline at the 3 end. Northern blot hybridization and reverse transcriptase PCR analyses showed that TcTREZO transcripts are expressed as oligo(A)-terminated transcripts whose length corresponds to the unit size of the element (1.6 kb). Transcripts of ϳ0.2 kb derived from a small part of TcTREZO are also detected in steady-state RNA. TcTREZO transcripts are unspliced and not translated. The copy number of TcTREZO sequences was estimated to be ϳ173 copies per haploid genome. TcTREZO appears to have been assembled by insertions of sequences into a progenitor element. Once associated with each other, these subunits were amplified as a new transposable element. TcTREZO shows site specificity for insertion, suggesting that a sequence-specific endonuclease could be responsible for its insertion at a unique site.

PLoS ONE, 2011

Background: The Trypanosoma cruzi genome was sequenced from a hybrid strain (CL Brener). However,... more Background: The Trypanosoma cruzi genome was sequenced from a hybrid strain (CL Brener). However, high allelic variation and the repetitive nature of the genome have prevented the complete linear sequence of chromosomes being determined. Determining the full complement of chromosomes and establishing syntenic groups will be important in defining the structure of T. cruzi chromosomes. A large amount of information is now available for T. cruzi and Trypanosoma brucei, providing the opportunity to compare and describe the overall patterns of chromosomal evolution in these parasites.

Infection and Immunity, 2007

A member of the Trypanosoma cruzi gp82 family, expressed on metacyclic trypomastigote surface and... more A member of the Trypanosoma cruzi gp82 family, expressed on metacyclic trypomastigote surface and identified by monoclonal antibody (MAb) 3F6, plays a key role in host cell invasion. Apart from the gp82 defined by MAb 3F6, no information is available on members of this protein family. From cDNA clones encoding gp82 proteins sharing 59.1% sequence identity, we produced the recombinant

PLoS ONE, 2011

Background: Trypanosoma cruzi has a single flagellum attached to the cell body by a network of sp... more Background: Trypanosoma cruzi has a single flagellum attached to the cell body by a network of specialized cytoskeletal and membranous connections called the flagellum attachment zone. Previously, we isolated a DNA fragment (clone H49) which encodes tandemly arranged repeats of 68 amino acids associated with a high molecular weight cytoskeletal protein.

International Journal for Parasitology, 2014

The identification of new targets for vaccine and drug development for the treatment of Chagas&am... more The identification of new targets for vaccine and drug development for the treatment of Chagas' disease is dependent on deepening our understanding of the parasite genome. Vectors for genetic manipulation in Trypanosoma cruzi basically include those that remain as circular episomes and those that integrate into the parasite's genome. Artificial chromosomes are alternative vectors to overcome problematic transgene expression often occurring with conventional vectors in this parasite. We have constructed a series of vectors named pTACs (Trypanosome Artificial Chromosomes), all of them carrying telomeric and subtelomeric sequences and genes conferring resistance to different selection drugs. In addition, one pTAC harbours a modified GFP gene (pTAC-gfp), and another one carries the ornithine decarboxilase gene from Crithidia fasciculata (pTAC-odc). We have encountered artificial chromosomes generated from pTACs in transformed T. cruzi epimastigotes for every version of the designed vectors. These extragenomic elements, in approximately 6-8 copies per cell, remained as linear episomes, contained telomeres and persisted after 150 and 60 generations with or without selection drugs, respectively. The linear molecules remained stable through the different T. cruzi developmental forms. Furthermore, derived artificial chromosomes from pTAC-odc could complement the auxotrophy of T. cruzi for polyamines. Our results show that pTACs constitute useful tools for reverse functional genetics in T. cruzi that will contribute to a better understanding of T. cruzi biology.

Infection and Immunity, 2007

A member of the Trypanosoma cruzi gp82 family, expressed on metacyclic trypomastigote surface and... more A member of the Trypanosoma cruzi gp82 family, expressed on metacyclic trypomastigote surface and identified by monoclonal antibody (MAb) 3F6, plays a key role in host cell invasion. Apart from the gp82 defined by MAb 3F6, no information is available on members of this protein family. From cDNA clones encoding gp82 proteins sharing 59.1% sequence identity, we produced the recombinant proteins J18 and C03, the former containing and the latter lacking the epitope for MAb 3F6. Polyclonal antibodies to J18 and C03 proteins were generated and used, along with MAb 3F6, to analyze the expression and cellular localization of gp82 family members in metacyclic forms of CL and G strains, which belong to highly divergent genetic groups. By two-dimensional gel electrophoresis and immunoblotting, molecules of 82 to 86 kDa, focusing at pH 4.6 to 5.4, and molecules of 72 to 88 kDa, focusing at pH 4.9 to 5.7, were visualized in CL and G strains, respectively. Flow cytometry and microscopic analysis revealed in both strains similar expression of MAb 3F6-reactive gp82 in live and permeabilized parasites, indicating its surface localization. The reaction of live parasites of both strains with anti-J18 antibodies was weaker than with MAb 3F6 and was increased by permeabilization. Anti-C03 antibodies bound predominantly to flagellar components in permeabilized G strain parasites, but in the CL strain the flagellum was not the preferential target for these antibodies. Host cell invasion of metacyclic forms was inhibited by J18 protein, as well as by MAb 3F6 and anti-J18 antibodies, but not by C03 protein or anti-C03 antibodies.

Gene, 2003

We present a useful refinement of the molecular karyotype of clone CL Brener, the reference clone... more We present a useful refinement of the molecular karyotype of clone CL Brener, the reference clone of the Trypanosoma cruzi Genome Project. The assignment of 210 genetic markers (142 expressed sequence tags (ESTs), seven cDNAs, 32 protein-coding genes, eight sequence tagged sites (STSs), 21 repetitive sequences) to the chromosomal bands separated by pulsed field gel electrophoresis (PFGE) identified 61 chromosome-specific markers, two size-polymorphic chromosomes and seven linkage groups. Fourteen new repetitive elements were isolated in this work and mapped to the chromosomal bands. We found that at least ten repetitive elements can be mapped to each chromosomal band, which may render the whole genome sequence assembly a difficult task. To construct the integrated map of chromosomal band XX, we used yeast artificial chromosome (YAC) overlapping clones and a variety of probes (i.e. known gene sequences, ESTs, STSs generated from the YAC ends). The total length covered by the YAC contig was approximately 1.3 Mb, covering 37% of the entire chromosome. We found some degree of polymorphism among YACs derived from band XX. These results are in agreement with data from phylogenetic analysis of T. cruzi which suggest that clone CL Brener is a hybrid genotype [Mol. Biochem. Parasitol. 92 (1998) 253; Proc. Natl. Acad. Sci. USA 98 ]. The physical map of the chromosomal bands, together with the isolation of specific chromosomal markers, will contribute in the global effort to sequence the nuclear genome of this parasite. q

Eukaryotic Cell, 2007

A new family of site-specific repeated elements identified in Trypanosoma cruzi, which we named T... more A new family of site-specific repeated elements identified in Trypanosoma cruzi, which we named TcTREZO, is described here. TcTREZO appears to be a composite repeated element, since three subregions may be defined within it on the basis of sequence similarities with other T. cruzi sequences. Analysis of the distribution of TcTREZO in the genome clearly indicates that it displays site specificity for insertion. Most TcTREZO elements are flanked by conserved sequences. There is a highly conserved 68-bp sequence at the 5 end of the element and a sequence domain of ϳ500 bp without a well-defined borderline at the 3 end. Northern blot hybridization and reverse transcriptase PCR analyses showed that TcTREZO transcripts are expressed as oligo(A)-terminated transcripts whose length corresponds to the unit size of the element (1.6 kb). Transcripts of ϳ0.2 kb derived from a small part of TcTREZO are also detected in steady-state RNA. TcTREZO transcripts are unspliced and not translated. The copy number of TcTREZO sequences was estimated to be ϳ173 copies per haploid genome. TcTREZO appears to have been assembled by insertions of sequences into a progenitor element. Once associated with each other, these subunits were amplified as a new transposable element. TcTREZO shows site specificity for insertion, suggesting that a sequence-specific endonuclease could be responsible for its insertion at a unique site.

PLoS ONE, 2011

Background: The Trypanosoma cruzi genome was sequenced from a hybrid strain (CL Brener). However,... more Background: The Trypanosoma cruzi genome was sequenced from a hybrid strain (CL Brener). However, high allelic variation and the repetitive nature of the genome have prevented the complete linear sequence of chromosomes being determined. Determining the full complement of chromosomes and establishing syntenic groups will be important in defining the structure of T. cruzi chromosomes. A large amount of information is now available for T. cruzi and Trypanosoma brucei, providing the opportunity to compare and describe the overall patterns of chromosomal evolution in these parasites.