ABSTRACT
Strategies to construct the physical map of the Trypanosoma cruzi nuclear genome have to capitalize on three main advantages of the parasite genome, namely (a) its small size, (b) the fact that all chromosomes can be defined, and many of them can be isolated by pulse field gel electrophoresis, and (c) the fact that simple Southern blots of electrophoretic karyotypes can be used to map sequence tagged sites and expressed sequence tags to chromosomal bands. A major drawback to cope with is the complexity of T. cruzi genetics, that hinders the construction of a comprehensive genetic map. As a first step towards physical mapping, we report the construction and partial characterization of a T. cruzi CL-Brener genomic library in yeast artificial chromosomes (YACs) that consists of 2,770 individual YACs with a mean insert size of 365 kb encompassing around 10 genomic equivalents. Two libraries in bacterial artificial chromosomes (BACs) have been constructed, BACI and BACII. Both libraries represent about three genome equivalents. A third BAC library (BAC III) is being constructed. YACs and BACs are invaluable tools for physical mapping. More generally, they have to be considered as a common resource for research in Chagas disease.
Subject(s)
Chromosome Mapping , Chromosomes, Artificial, Yeast , Chromosomes, Bacterial/genetics , Cloning, Organism , Genome, Protozoan , Trypanosoma cruzi/genetics , Animals , Genomic LibraryABSTRACT
Eleven antigenic clones were isolated from a Trypanosoma cruzi cDNA expression library using a pool of Chagasic sera from Venezuelan patients. These clones were tested with 26 Chagasic sera and 11 sera from patients with other diseases. One clone (Clone 2) reacted with the majority of the Chagasic sera and did not react with the non-Chagasic sera. Restriction enzyme digestion of Clone 2 DNA showed that it contained an insert of 5.2 kb. In a Southern blot of a pulse field gel electrophoresis, Clone 2 hybridized to three T. cruzi chromosomal fragments. By Northern blot analyses, it was observed that the clone hybridizes to a major T. cruzi RNA band of 0.97 kb and to two minor bands of 4.8 and 6.3 kb. The expression of these three RNAs was higher in trypomastigotes than in epimastigotes or spheromastigotes. Specific antibodies isolated against the beta-galactosidase-Clone 2 fusion protein expressed in E. coli reacted with a 28 kilodalton protein in T. cruzi.
Subject(s)
Antigens, Protozoan/isolation & purification , Protozoan Proteins/isolation & purification , Trypanosoma cruzi/immunology , Animals , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Chagas Disease/immunology , Cloning, Molecular , DNA, Complementary/genetics , DNA, Protozoan/genetics , Gene Expression Regulation , Genes, Protozoan , Humans , Molecular Weight , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Recombinant Proteins/immunology , Sensitivity and Specificity , Trypanosoma cruzi/geneticsABSTRACT
A survey of human Trypanosoma cruzi infection in four rural communities of two Venezuelan states with different epidemiological Chagas' disease situations was carried out using the Dot-ELISA and conventional serology. In the two hamlets of Zulia state, no seropositives were found in the under-15 age group whereas seropositivity in the over-15 group was 15.6%. In Cojedes state, the two hamlets studied exhibited a seropositivity of 8.9% in the under-15 group and 51.6% in the over-15 group. Upon comparison with conventional methods, Dot-ELISA evidenced high co-positivity, co-negativity and efficiency indexes. In the samples taken from Zulia, the predictive value of the test was 66% and 60% for cytoplasmatic and integral antigens, respectively; with the Cojedes samples, 100% and 95%. The results suggest that Dot-ELISA could be a practical alternative for seroepidemiological Chagas' disease studies in underdeveloped regions.