Sequence and Functional Analysis of the Intergenic Regions Separating Babesial Rhoptry-Associated Protein-1 (rap-1)Genes

Abstract

Suarez, C. E., Palmer, G. H., Hötzel, I., Hines, S. A., and McElwain, T. F. 1998. Sequence and functional analysis of the intergenic regions separating babesial rhoptry-associated protein-1 (rap-1) genes.Experimental Parasitology90, 189–194. The rhoptry-associated protein 1 (RAP-1) expressed by all babesial parasites is encoded by tandemly arranged genes separated by discrete intergenic (IG) regions. We hypothesize that these IG regions regulaterap-1gene expression. InBabesia bovistwo identicalrap-1gene copies are separated by a 1.0-kb noncoding region which is also exactly conserved 5′ to therap-1gene 1. In contrast, the complexB. bigemina rap-1locus contains at least 5 polymorphicrap-1agenes separated by uncharacterized 3.38-kb regions. A genomic clone encoding the 3′ sequence ofrap-1gene copy 1, the 1 kb IG region, and the 5′ sequence of gene copy 2 was obtained by PCR amplification of DNA from the Mo7 biological clone ofB. bovisand sequenced. This was follow by amplification and sequence analysis of the 3.38-kb region separating twoB. bigemina rap-1agenes, revealing the presence of two different IG regions denominated IG-1 (0.7 kb) and IG-2 (1.3 kb), flanking a newly identifiedrap-1borf. Sequence analysis and comparison among babesialrap-1IG regions fromB. bovis, B. bigemina, B. canis, andB. ovisrevealed conservation of at least three putative regulatory boxes consistently positioned 5′ of the start of therap-1orfs. To determine whetherrap-1IG regions contained a functional promoter, the entire 1-kb IG region fromB. boviswas cloned into pCAT, a promoterless plasmid containing thecatgene. The IG region in the 5′ → 3′ orientation strongly promoted transcriptionin vitroby homologousB. bovisRNA polymerases. The presence of conserved regions 5′ to eachrap-1gene copy and among other babesialrap-1IG regions and thein vitropromoter function in the 5′ → 3′ orientation support a role for the IG region inrap-1gene regulation.