High order structure in the Babesia bovis locus of active ves transcription

Babesia bovis is an intraerythrocytic protozoan which causes severe disease in cattle following the transmission by its vector tick. It can persist life-long in animal hosts which survive acute infection. Cytoadhesion of parasite-infected red blood cells (IRBC) to the endothelium of host deep organs, and periodic antigenic variation of the adhesive ligands on the infected erythrocyte surface are two major survival strategies. Previous work had identified the heterodimeric variant erythrocyte surface antigen 1 (VESA1), which is encoded by ves1alpha/beta gene pairs among the ves multigene family, as a key bifunctional virulence factor due to its involvement in both cytoadhesion and antigenic variation. In the B. bovis C9.1 clonal line, the locus of active ves transcription (LAT) of ves1alpha/beta gene pairs has been revealed, and segmental gene conversion has been demonstrated to contribute greatly to B. bovis antigenic variation. The unique gene organization of the LAT, which contains two closely juxtaposed, divergent ves1alpha/beta genes sharing overlapping 5'untranslated regions (UTR), also exists at many other non-expressed ves gene loci. It is unknown how the LAT acquires its distinctive active transcription state among ves genes. To determine whether stable, higher order structure exists at the LAT which may be related to its active transcription, in vivo 3-dimensional DNA structural characteristics were examined initially through a combination of structure-protective genomic DNA extraction and DNA two-Dimensional (2-D) gel electrophoresis. In vivo psoralen crosslinking was also tried to stabilize LAT-associated higher-order DNA structure. Some higher-order DNA structures were detected with both structure-preserving methods and could most likely be associated with the LAT regarding active transcription although other evidences are essential to substantially prove and interpret their significance. In addition, an in vitro study also supported the potential of ves intergenic regions (IGr) to form higher-order structures when induced by thermal remodeling (TR). Several nucleases were used to further investigate the chromatin structural characteristics of ves genes. Several locus-specific nucleosomal repeat lengths were estimated found indistinguishable from bulk chromatin. In contrast, the LAT adopts an unusual chromatin structure featuring 5 MNase-hypersensitive sites (MH sites) in the IGr rather than three, and two protected IGr-flanking regions. The rates of hybridization signal losses from different gene loci upon the treatment with different concentrations of MNase or DNase I were estimated. Both enzymes produced three highly repeatable patterns of ves nuclease sensitivity. The LAT is significantly more sensitive than are some non-expressed ves genes or even the active B. bovis beta-tubulin and glyceraldehydes-3-phosphate dehydrogenase (GADPH) genes, consistent with their active state. In contrast, among non-expressed ves genes two populations may be defined: genes which are relatively insensitive to digestion, and those which are intermediate insensitivity between the LAT and the insensitive ves loci. All those facts suggested that different chromatin structures explain the differential transcriptional status among ves genes, which could be mediated by differential epigenetic modifications such as acetylation/methylation of histone H3.