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

Abstract

We have initiated large-scale sequencing of the third smallest chromosome of the CL Brener strain of Trypanosoma cruzi and we report here the complete sequence of a contig consisting of three cosmids. This contig covers 93.4 kb and has been found to contain 20-30 novel genes and several repeat elements, including a novel chromosome 3-specific 400-bp repeat sequence. The intergenic sequences were found to be rich in di-and trinucleotide repeats of varying lengths and also contained several known T. cruzi repeat elements. The sequence contains 29 open reading frames (ORFs) longer than 700 bp, the longest being 5157 bp, and a large number of shorter ORFs. Of the long ORFs, seven show homology to known genes in parasites and other organisms, whereas four ORFs were confirmed by sequencing of cDNA clones. Two shorter ORFs were confirmed by a database homology and a cDNA clone, respectively, and one RNA gene was identified. The identified genes include two copies of the gene for alanine-aminotransferase as well as genes for glucose-6-phosphate isomerase, protein kinases and phosphatases, and an ATP synthase subunit. An interesting feature of the sequence was that the genes appear to be organized in two long clusters containing multiple genes on the same strand. The two clusters are transcribed in opposite directions and they are separated by an ∼20-kb long, relatively GC-rich sequence, that contains two large repetitive elements as well as a pseudogene for cruzipain and a gene for U2snRNA. It is likely that this strand switch region contains one or more regulatory and promoter regions. The reported sequence provides the first insight into the genome organization of T. cruzi and shows the potential of this approach for rapid identification of novel genes.

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 fractional GC content for all the GSSs varied from 0.18-0.71, with ∼69% of the sequences falling in the 0.47-0.57 range.…”

“…Recently, the complete sequence of chromosome 1 from L. major Friedlin (Myler et al 1999) and a partial sequence of chromosome 3 from T. cruzi (Andersson et al 1998) were obtained, showing in both cases a similar organization with two clusters of genes per chromosome transcribed in opposite directions. These studies also showed that the gene density is high in the regions where genes are clustered, with ∼1 gene every 3.6 Kb in both cases.…”

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

“…Recently, Andersson et al (1998) reported the complete sequence of a 93.4 kb contig from T. cruzi chromosome 3. This contig contains a strand-switch region where genes appear to be organized in two long clusters harboring multiple genes on the same strand, with the two clusters oriented in opposite directions.…”

Gene Survey of the Pathogenic Protozoan Trypanosoma cruzi

“…cruzi can be estimated to have ∼12,000 genes from the gene density of about 1 gene per 3.5-4 kb, as revealed by genomic sequencing (Andersson et al 1998) and the haploid genome size of ∼45 Mb in the T. cruzi reference clone CL Brener (J. Swindle, unpubl.). Because a considerable part of the T. cruzi genome comprises several large gene families as well as other repeat sequences (Requena et al 1996), this number of genes should be an overestimate.…”

“…Neither vaccines nor safe and efficient drug treatment are presently available against this debilitating disease. The genome project for T. cruzi involves both genomic sequencing (Andersson et al 1998) and identification of functional genes through generation of expressed sequence tags (ESTs) (Brandão et al 1997;Verdun et al 1998), a cost-effective technique in gene discovery (Venter 1993;Okubo and Matsubara 1997). The ongoing parasite genome projects of the three kinetoplastids, Leishmania major (Blackwell 1997), Trypanosoma brucei (Melville 1997), and T. cruzi (Zingales et al 1997), are critical in identifying orthologous genes involved in mechanisms among kinetoplastids.…”