Unusual chromatin structure associated with monoparalogous transcription of the Babesia bovis ves multigene family

Abstract

Rapid antigenic variation in Babesia bovis involves the variant erythrocyte surface antigen-1 (VESA1), a heterodimeric protein with subunits encoded by two branches of the ves multigene family. The ves1α and ves1β gene pair encoding VESA1a and 1b, respectively, are transcribed in a monoparalogous manner from a single locus of active ves transcription (LAT), just one of many quasi-palindromic ves loci. To determine whether this organization plays a role in transcriptional regulation, chromatin structure was first assessed. Limited treatment of isolated nuclei with micrococcal nuclease to assay nucleosomal patterning revealed a periodicity of 156–159 bp in both bulk chromatin and specific gene coding regions. This pattern also was maintained in the intergenic regions (IGr) of non-transcribed ves genes. In contrast, the LAT IGr adopts a unique pattern, yielding an apparent cluster of five closely-spaced hypersensitive sites flanked by regions of reduced nucleosomal occupancy. ves loci fall into three patterns of overall sensitivity to micrococcal nuclease or DNase I digestion, with only the LAT being consistently very sensitive. Non-transcribed ves genes are inconsistent in their sensitivity to the two enzymatic probes. Nonlinear DNA structure in chromatin was investigated to determine whether unique structure arising as a result of the quasi-palindromic nature of the LAT may effect transcriptional control. The in vitro capacity of ves IGr sequences to adopt stable higher-order DNA structure is demonstrated here, but the presence of such structure in vivo was not supported. Based upon these results a working model is proposed for the chromatin structural remodeling responsible for the sequential expression of ves multigene family members from divergently-organized loci.

Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform

“…Digestion products were probed by staining with SybrGreen to detect total chromatin, and by Southern blotting to detect BbAcC3_Bb rad51 -derived sequences. BbAcC3_Bbrad51wt sequences provided a ladder of digestion products with a nucleosomal periodicity of approximately 157–158 bp, matching that of total native chromatin (Fig 7A) and previously reported values [44], indicative of assembly on nucleosomes. Southern blot probing of isolated plasmid or genomic DNAs, either uncut or cut with informative restriction endonucleases, revealed the expansion of both ends of established BbAcC3 and BbAcC3_Bb rad51 chromosomes (Fig 7B).…”

Knockout of Babesia bovis rad51 ortholog and its complementation by expression from the BbACc3 artificial chromosome platform

“…BbAcC3_Bbrad51wt sequences provided a ladder of digestion products with a nucleosomal periodicity of approximately 157-158 bp, matching that of total native chromatin (Fig. 7A) and previously reported values [37], indicative of assembly on nucleosomes. Southern blot probing of isolated plasmid or genomic DNAs, either uncut or cut with informative restriction endonucleases, revealed the expansion of both ends of established BbAcC3 and BbAcC3_Bb rad51 chromosomes, indicative of telomere lengthening (Fig.…”

“…Lanes are: (1) PstI-cut purified DNA, (lane 2) chromatin partially-digested with 0 U, (lane 3) 0.0075 U, (lane 4) 0.015 U, (lane 5) 0.03 U, or (lane 6) 0.06 U of MNase prior to isolation of DNA. Asterisks demonstrate correspondence among the nucleosomal patterns generated from the samples, which form a ladder with 156-159 bp spacing between bands, as previously reported [37]. (center panel) Southern blot of the same gel as in the left panel, probed with β-lactamase sequences to detect the pBluescript backbone of BbACc3_Bb rad51 wt.…”

“…Probes were labeled with 11-dUTP-digoxigenin, following manufacturer’s instructions, and detected as described above. To observe assembly of BbACc3 and BbACc3_Bbrad51wt into chromatin, nuclei were isolated from transformed parasites, the chromatin partially digested with micrococcal nuclease (MNase), and the fragments isolated as described [37]. To observe overall nucleosomal assembly and spacing, digestion products were stained in-gel with SybrGold.…”

Large, rapidly evolving gene families are at the forefront of host–parasite interactions inApicomplexa

“…Expressed genes are found in a tandem configuration and are expressed from LATs by a bidirectional promoter. It is thought that chromatin remodelling is required for transcription suggesting there may be some commonalities with control of var expression (Huang et al 2013 ). However, it is not clear that chromatin remodelling plays a direct role in monoallelic expression.…”