Roberto Hernández

PRIBOTEX EXPRESSION VECTOR : A PTEX DERIVATIVE FOR A RAPID SELECTION OF TRYPANOSOMA CRUZI TRANSFECTANTS

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-cat expressed a chloramphenicol (Cm) acetyltransferase (CAT) activity 16,000-fold greater than the activity observed with the parental vector pTEX-cat. Moreover, T. cruzi cells transformed with pRIBOTEX and pRIBOTEX-cat exhibited logarithmic growth in the presence of Geneticin (G418) 2 weeks earlier than that observed with controls transformed with pTEX. The plasmid copy number in stably transformed trypanosomes was about 50-times higher in cultures transformed with pTEX-cat than in cells transformed with pRIBOTEX or pRIBOTEX-cat. However, the neo RNA steady-state level and the CAT activity observed among the stably transfected cultures showed only modest differences. Finally, it was found that the pRIBOTEX vector was not episomally maintained as pTEX, but integrated into a chromosome indistinguishable from the one encoding rRNA. These features make pRIBOTEX a useful tool for transfection and rapid expression of genes in T. cruzi.

Ribosomal RNA genes in eukaryotic microorganisms: witnesses of phylogeny?

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. However, it is likely that these organisms have preserved variations in the organization of a versatile gene that may be seen as living records of evolution. Here, we review the organization of the main rRNA transcription unit (rDNA) and the 5S rRNA genes (5S rDNA). These genes are reiterated in the genome of microbial eukaryotes and may be coded alone, in tandem repeats, linked to each other or linked to other genes. They may be found in the chromosome or extrachromosomally in linear or circular units. rDNA coding regions may contain introns, sequence insertions, protein-coding genes or additional spacers. The 5S rDNA can be found in tandem repeats or genetically linked to genes transcribed by RNA polymerases I, II or III. Available information from about a hundred microbial eukaryotes was used to review the unexpected diversity in the genomic organization of rRNA genes.

Trypanosoma cruzi ribosomal RNA: internal break in the large-molecular-mass species and number of genes.

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 cleavage during in vitro manipulations. Calculations of mass, from gel electrophoretograms, for the large and small main ribosomal ribonucleic acid species and for the two chains derived from the large species gave values of 1.37, 0.84, 0.70 and 0.57 X 10(6) daltons, respectively. Sedimentation velocity measurements in sucrose gradients and in the analytical ultracentrifuge indicated sedimentation coefficients of 24 and 18 S for the large and small main species, respectively. Saturation hybridization curves showed that the nuclear genome, quantified by chemical analysis, contains about 114 ribosomal ribonucleic acid gene copies.

Primary structure of Trypanosoma cruzi small-subunit ribosomal RNA coding region: comparison with other trypanosomatids

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 nucleotides in the T. cruzi gene occur in highly variable regions of rRNA genes. A phylogenetic analysis was performed with the SSU rRNA sequences from these four trypanosomatids and from Euglena gracilis as an outgroup. The Leishmania and Crithidia sequences were remarkably similar to each other (not separable statistically). Given the standard errors associated with the branching order of the two Trypanosoma species and the Leishmania-Crithidia branch, the actual topology cannot be unequivocally determined using only the ribosomal sequences analyzed here.

Biological characterization and genetic diversity of Mexican isolates of Trypanosoma cruzi

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 trypomastigotes, infectivity to, and in vitro killing of cultured Vero and P388 cells. Isoenzyme patterns of 11 enzymatic systems and 16 loci were also determined for the Mexican isolates. The parasites showed differences in growth, depending if they were cultured in LIT with hemin or in Graces media. Transformation was obtained only in Graces medium and differences were observed between the stocks. Stocks Z10 and Z21 showed the highest percentage of transformation within the Mexican isolates (39 and 41%, respectively). A second group showed percentages of transformation between 15 and 28%. In contrast, the South American strains showed higher rates of transformation (36-65%). Infection of cultured cells by isolates Z10 and H5 was evaluated in both Vero and P388 cells. Differences were observed both in the percentage of infected cells as well as in the number of amastigotes per cell. Differences in the ability to cause in vitro killing of P388 cells were also observed among the isolates. Isoenzyme analysis revealed genetic variation between the isolates, each of them with an unique zymodeme. This genetic analysis revealed, in general, a clustering based on the geographical origin of the isolates. Finally, correlation with clinical symptoms is discussed.

Genotype and virulence correlation within Mexican stocks of Trypanosoma cruzi isolated from patients

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 restriction fragment length polymorphism associated with a repetitive element from the rRNA gene spacer was performed. No differences in in vitro growth or in parasitic load in vivo were found among the stocks. While three stocks showed low virulence for mice, the other two stocks killed 80 and 100% of the infected mice. In addition, Southern blot of total DNA hybridized with a repetitive element from the rRNA gene spacer showed two clearly distinct patterns that correlated with the observed ability of the stocks to kill infected mice. Our results show a correlation among the ability to kill BALB/c mice, the genetic pattern and clinical symptoms produced by the different stocks in the infected patients.

Trypanosoma cruzi ribosomal DNA: mapping of a putative distal promoter

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 from another T. cruzi strain [Dietrich et al., Gene 125 (1993) 103-107]. Sequence comparisons of the nt flanking both T. cruzi tsp with the homologous regions from both other trypanosomatids, and other eukaryotes, indicate that these sequences are poorly conserved within the family Trypanosomatidae. This finding reinforces the proposal that the speciation of trypanosomatids may have occurred early in evolution.

Cloning and sequencing of two actin genes from Taenia solium (Cestoda)

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. solium expresses an actin transcript of about 1,400 bases and a protein of 45,000 Da.

Chagas’ Disease: Pregnancy and Congenital Transmission

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 control transmission, it is essential to test all pregnant women living in endemic countries and all pregnant women having migrated from, or having lived in, endemic countries. All children born to seropositive mothers should be tested not only within the first month of life but also at ~6 months and ~12 months of age. The diagnosis is made by identification of the parasite in blood before the age of 6 months and by identification of the parasite in blood and/or positive serology after 10 months of age. Follow up for a year is essential as a significant proportion of cases are initially negative and are only detected at a later stage. If the condition is diagnosed and treated early, the clinical response is excellent and the majority of cases are cured.

Trypanosoma cruzi: Multiple actin isovariants are observed along different developmental stages

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 subcellular distribution of actin among the developmental stages were identified. The existence of five actin variants in each developmental stage with distinct patterns of expression were uncovered by immunoblotting of protein extracts separated 2D-SDS gels. The isoelectric points of the actin variants in epimastigotes ranged from 4.45 to 4.9, whereas they ranged from 4.9 to 5.24 in trypomastigotes and amastigotes. To determine if the actin variants found could represent previously unidentified actins, we performed a genomic survey of the T.cruzi GeneDB database and found 12 independent loci encoding for a diverse group of actins and actin-like proteins that are conserved among trypanosomatids.