Selective generation of chromosomal cosmid libraries within the Trypanosoma cruzi genome project

Abstract

From a total genomic cosmid library of the pathogen Trypanosoma cruzi, specific sublibraries of the smallest four chromosomes were isolated by hybridization of the respective chromosomal bands obtained from pulsed-field gels. These libraries form the basis for initial mapping analyses that should provide information useful for both the ongoing physical mapping of the entire genome and eventual sequence analyses. Selectivity of the procedure was high with 75% to 92%, although cross-hybridization had to be expected from ubiquitous DNA features, such as centromeric and telomeric sequences, and other regions homologous between individual chromosomes. Overall, the number of identified clones was slightly higher than expected but well within the intrinsic experimental variation considering the uncertainty about the exact genome size, the variability in clonability and the higher frequency of repeat sequences in larger chromosomes. Chromosome III- and IV-specific cosmids were analyzed on Southern blots of chromosomal separations. For strain CL Brener, all clones tested exhibited cross-hybridization to a homologous chromosome larger than 1 Mbp, supporting the assumption of the respective chromosome couple being diploid pairs.

Physical Mapping of a 670-kb Region of Chromosomes XVI and XVII from the Human Protozoan Parasite Trypanosoma cruzi Encompassing the Genes for Two Immunodominant Antigens

“…This hybridization pattern corroborates the hypothesis that bands XVI and XVII carry size-polymorphic homologous chromosomes (Table 1). The existence of two homologs of a diploid chromosome that differ in size had been reported previously in T. cruzi and Trypanosoma brucei (Turner et al 1997;Frohme et al 1998;Hanke et al 1998;Melville et al 1998).…”

“…Classical genetic studies on this parasite have been hampered by the existence of many parasite strains with distinct biological and immunological characteristics, the lack of sexual stages, and a high variability in both number and size of chromosomes (Brener 1973;Solari 1995;Cano et al 1995;Henriksson et al 1995Henriksson et al , 1996Santos et al 1997). For this reason, one of the main goals of the recently started T. cruzi Genome Initiative is to construct a physical map of the parasite's genome as a resource for DNA sequence analysis (Hanke et al 1996Zingales et al 1997a;Ferrari et al 1997;Frohme et al 1998). A physical map of the T. cruzi genome would also facilitate the identification and characterization of genetic loci.…”

A Random Sequencing Approach for the Analysis of the Trypanosoma cruzi Genome: General Structure, Large Gene and Repetitive DNA Families, and Gene Discovery

“…These GSS (Genomic Sequence Survey) sequences represent 4.3 Mb of readable sequences or ∼10% of the parasite haploid genome, which is ∼40 Mb (Frohme et al 1998). The total GC content of the sequence produced was 50.9%, a value that is slightly larger than the one obtained from estimations made on 8796 T. cruzi ESTs (49.6 %), and the one obtained for 93.4 Kb from chromosome 3 (48.5%) (Andersson et al 1998).…”

“…The estimation also included the size (GS) of the gene/sequence element to account for the fact that bigger sequences should be more represented in a random sequence survey than smaller ones at the same copy number. For this calculation the haploid genome size (HGS) used was 4 ‫ן‬ 10 7 bp (Frohme et al 1998) and the total number of GSS (TGSS) was 11,459:…”

Complete Sequence of a 93.4-kb Contig from Chromosome 3 of Trypanosoma cruzi Containing a Strand-Switch Region

“…The cosmids were selected from a sublibrary enriched for cosmids containing inserts from chromosome 3 and its homolog (Frohme et al 1998). The first cosmid (1o17) was chosen as a starting cosmid for a region of the chromosome and the following two cosmids (1b21 and 1m17) were selected on the basis of confirmed overlaps to the already sequenced cosmids.…”