Organization, transcription, and expression of rhoptry associated protein genes in the Babesia bigemina rap-1 locus.

The Babesia bigemina rap-1 gene locus contains five tandemly arranged copies of rap-1a genes. However, the size of the locus, as defined by conserved, unrelated orfs at the 5' and 3' ends, suggests that additional genes may be present. In this study, we identified all additional genes in the locus and characterized their pattern of expression in merozoites. The rap-1a genes are separated by 3.38-kbp intergenic (IG) regions, each of which contains an identical copy of a related gene designated rap-1b. One additional copy of rap-1b and one copy of another related gene designated rap-1c is present in the 3' end of the locus. Common sequence features that define the Babesia rap-1 family are present in rap-1b and rap-1c, but otherwise these genes average only 27% identity to rap-1a. Homologues of the rap-1b and rap-1c genes identified in diverse B. bigemina strains have a high degree of predicted amino acid sequence conservation (averaging >90%), with the largest number of changes in the carboxyl end of RAP-1c. We tested whether all rap-1 genes in the locus are co-transcribed in merozoites using RT-PCR, Northern blots, and quantitative real-time PCR. Rap-1a genes produce the most abundant transcripts of the family, while rap-1b transcripts are the least abundant despite the large number of gene copies. Similar patterns of transcription were observed whether merozoites were obtained from in vitro cultures or in vivo infection. Immunoblot analysis of merozoites revealed the expected RAP-1a expression but failed to detect expressed RAP-1b and RAP-1c, indicating that expression of the rap-1 genes is regulated both at the transcriptional and translational levels.