Hemizygous subtelomeres of an African trypanosome chromosome may account for over 75% of chromosome length.

African trypanosomes are parasitic protozoa that infect a wide range of mammals, including humans. These parasites remain extracellular in the mammalian bloodstream, where antigenic variation allows them to survive the immune response. The Trypanosoma brucei nuclear genome sequence has been published recently. However, the significant chromosome size polymorphism observed among strains and subspecies of T. brucei, where total DNA content may vary up to 30%, necessitates a comparative study to determine the underlying basis and significance of such variation between parasites. In addition, the sequenced strain (Tb927) presents one of the smallest genomes analyzed among T. brucei isolates; therefore, establishing polymorphic regions will provide essential complementary information to the sequencing project. We have developed a Tb927 high-resolution DNA microarray to study DNA content variation along chromosome I, one of the most size-variable chromosomes, in different strains and subspecies of T. brucei. Results show considerable copy number polymorphism, especially at subtelomeres, but are insufficient to explain the observed size difference. Additional sequencing reveals that >50% of a larger chromosome I consists of arrays of variant surface glycoprotein genes (VSGs), involved in avoidance of acquired immunity. In total, the subtelomeres appear to be three times larger than the diploid core. These results reveal that trypanosomes can utilize subtelomeres for amplification and divergence of gene families to such a remarkable extent that they may constitute most of a chromosome, and that the VSG repertoire may be even larger than reported to date. Further experimentation is required to determine if these results are applicable to all size-variable chromosomes.

Separation, digestion, and cloning of intact parasite chromosomes embedded in agarose.

The chromosomes of most protozoan parasites cannot be visualized using conventional microscopy because they are too small and do not condense sufficiently at metaphase. Therefore, the development of pulsed field gel electrophoresis allowed the resolution of many parasite karyotypes for the first time. The ability to prepare intact chromosomes in agarose plugs and to isolate individual homologs by electrophoresis has led to many new applications in parasite genomic analysis. This chapter describes the preparation of chromosome plugs from single-celled protozoan parasites, providing numerous tips on how to achieve the highest-quality preparations that will last for years. We also provide detailed protocols for the manipulation of individual excised chromosomes, including restriction mapping and preparation of chromosome shotgun libraries as used in many of the genomic sequencing projects. The protocols provided here underpin several of the advanced methods of genomic analysis and manipulation described in this volume of parasite genomics protocols.

The sequence and analysis of Trypanosoma brucei chromosome II.

We report here the sequence of chromosome II from Trypanosoma brucei, the causative agent of African sleeping sickness. The 1.2-Mb pairs encode about 470 predicted genes organised in 17 directional clusters on either strand, the largest cluster of which has 92 genes lined up over a 284-kb region. An analysis of the GC skew reveals strand compositional asymmetries that coincide with the distribution of protein-coding genes, suggesting these asymmetries may be the result of transcription-coupled repair on coding versus non-coding strand. A 5-cM genetic map of the chromosome reveals recombinational 'hot' and 'cold' regions, the latter of which is predicted to include the putative centromere. One end of the chromosome consists of a 250-kb region almost exclusively composed of RHS (pseudo)genes that belong to a newly characterised multigene family containing a hot spot of insertion for retroelements. Interspersed with the RHS genes are a few copies of truncated RNA polymerase pseudogenes as well as expression site associated (pseudo)genes (ESAGs) 3 and 4, and 76 bp repeats. These features are reminiscent of a vestigial variant surface glycoprotein (VSG) gene expression site. The other end of the chromosome contains a 30-kb array of VSG genes, the majority of which are pseudogenes, suggesting that this region may be a site for modular de novo construction of VSG gene diversity during transposition/gene conversion events.

The DNA sequence of chromosome I of an African trypanosome: gene content, chromosome organisation, recombination and polymorphism.

The African trypanosome, Trypanosoma brucei, causes sleeping sickness in humans in sub-Saharan Africa. Here we report the sequence and analysis of the 1.1 Mb chromosome I, which encodes approximately 400 predicted genes organised into directional clusters, of which more than 100 are located in the largest cluster of 250 kb. A 160-kb region consists primarily of three gene families of unknown function, one of which contains a hotspot for retroelement insertion. We also identify five novel gene families. Indeed, almost 20% of predicted genes are members of families. In some cases, tandemly arrayed genes are 99-100% identical, suggesting an active process of amplification and gene conversion. One end of the chromosome consists of a putative bloodstream-form variant surface glycoprotein (VSG) gene expression site that appears truncated and degenerate. The other chromosome end carries VSG and expression site-associated genes and pseudogenes over 50 kb of subtelomeric sequence where, unusually, the telomere-proximal VSG gene is oriented away from the telomere. Our analysis includes the cataloguing of minor genetic variations between the chromosome I homologues and an estimate of crossing-over frequency during genetic exchange. Genetic polymorphisms are exceptionally rare in sequences located within and around the strand-switches between several gene clusters.

A new, expressed multigene family containing a hot spot for insertion of retroelements is associated with polymorphic subtelomeric regions of Trypanosoma brucei.

We describe a novel gene family that forms clusters in subtelomeric regions of Trypanosoma brucei chromosomes and partially accounts for the observed clustering of retrotransposons. The ingi and ribosomal inserted mobile element (RIME) non-LTR retrotransposons share 250 bp at both extremities and are the most abundant putatively mobile elements, with about 500 copies per haploid genome. From cDNA clones and subsequently in the T. brucei genomic DNA databases, we identified 52 homologous gene and pseudogene sequences, 16 of which contain a RIME and/or ingi retrotransposon inserted at exactly the same relative position. Here these genes are called the RHS family, for retrotransposon hot spot. Comparison of the protein sequences encoded by RHS genes (21 copies) and pseudogenes (24 copies) revealed a conserved central region containing an ATP/GTP-binding motif and the RIME/ingi insertion site. The RHS proteins share between 13 and 96% identity, and six subfamilies, RHS1 to RHS6, can be defined on the basis of their divergent C-terminal domains. Immunofluorescence and Western blot analyses using RHS subfamily-specific immune sera show that RHS proteins are constitutively expressed and occur mainly in the nucleus. Analysis of Genome Survey Sequence databases indicated that the Trypanosoma brucei diploid genome contains about 280 RHS (pseudo)genes. Among the 52 identified RHS (pseudo)genes, 48 copies are in three RHS clusters located in subtelomeric regions of chromosomes Ia and II and adjacent to the active bloodstream form expression site in T. brucei strain TREU927/4 GUTat10.1. RHS genes comprise the remaining sequence of the size-polymorphic "repetitive region" described for T. brucei chromosome I, and a homologous gene family is present in the Trypanosoma cruzi genome.