SEQUENCE AND FUNCTIONAL-ANALYSIS OF THE INTERGENIC REGIONS SEPARATINGBABESIAL RHOPTRY-ASSOCIATED PROTEIN-1 (RAP-1) GENES

The rhoptry-associated protein 1 (rap-1) expressed by all babesial par asites is encoded by tandemly arranged genes separated by discrete int ergenic (IG) regions. We hypothesize that these IG regions regulate ra p-1 gene expression. In Babesia bovis two identical rap-1 gene copies are separated by a 1.0-kb noncoding region which is also exactly conse rved 5' to the rap-1 gene 1, In contrast, the complex B. bigemina rap- 1 locus contains at least 5 polymorphic rap-1a genes separated by unch aracterized 3.38-kb regions. A genomic clone encoding the 3' sequence of rap-1 gene copy I, the 1 kb IG region, and the 5' sequence of gene copy 2 was obtained by PCR amplification of DNA from the Mo7 biologica l clone of B. bovis and sequenced. This was follow by amplification an d sequence analysis of the 3.38-kb region separating two B. bigemina r ap-1a genes, revealing the presence of two different IG regions denomi nated IG-1 (0.7 kb) and IG-2 (1.3 kb), flanking a newly identified rap -1b orf. Sequence analysis and comparison among babesial rap-1 IG regi ons from B. bovis, B. bigemina, B. canis, and B. ovis revealed conserv ation of at least three putative regulatory boxes consistently positio ned 5' of the start of the rap-1 orfs. To determine whether rap1 IG re gions contained a functional promoter, the entire 1-kb IG region from B. bovis was cloned into pCAT, a promoterless plasmid containing the c at gene. The IG region in the 5' --> 3' orientation strongly promoted transcription in vitro by homologous B. bovis RNA polymerases. The pre sence of conserved regions 5' to each rap-1 gene copy and among other babesial rap-1 IG regions and the in vitro promoter function in the 5' --> 3' orientation support a role for the IG region in rap-l gene reg ulation. (C) 1998 Academic Press.