RESUMEN
Strategies to construct the physical map of the Trypanosoma cruzi nuclear genome have to capitalize on three main advantage 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.
Asunto(s)
Animales , Mapeo Cromosómico , Genoma de Protozoos , Trypanosoma cruzi/genética , Cromosomas Artificiales de Levadura , Células Clonales , Lugares Marcados de SecuenciaRESUMEN
Current YAC libraries are plagued by a high frequency of chimeras--that is, clones containing fragments from multiple genomic regions. Chimeras are thought to arise largely through recombination in the yeast host cell. If so, the use of recombination-deficient yeast strains, such as rad52 mutants, might be expected to alleviate the problem. Here, we report the construction of megabase-sized human YACs in the rad52 strain MHY5201 and the determination of their rate of chimerism by fluorescence in situ hybridization analysis. Examination of 48 YACs showed a rate of chimerism of at most 8%, whereas YACs constructed in the wildtype host AB1380 showed a rate of about 50%. These results show that it is possible to significantly decrease the rate of YAC chimerism through the use of appropriate yeast host strains.