Sequence and functional analysis of the intergenic regions separating babesial rhoptry-associated protein-1 (rap-1) genes.
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 regulate rap-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 conserved 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 uncharacterized 3.38-kb regions. A genomic clone encoding the 3' sequence of rap-1 gene 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 of B. bovis and sequenced. This was follow by amplification and sequence analysis of the 3.38-kb region separating two B. bigemina rap-1a genes, revealing the presence of two different IG regions denominated 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 regions from B. bovis, B. bigemina, B. canis, and B. ovis revealed conservation of at least three putative regulatory boxes consistently positioned 5' of the start of the rap-1 orfs. To determine whether rap-1 IG regions contained a functional promoter, the entire 1-kb IG region from B. bovis was cloned into pCAT, a promoterless plasmid containing the cat gene. The IG region in the 5' --> 3' orientation strongly promoted transcription in vitro by homologous B. bovis RNA polymerases. The presence 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-1 gene regulation.
Suarez
C E
CE
Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, 99164-7040, USA. ces@vetmed.wsu.edu
Palmer
G H
GH
Hötzel
I
I
Hines
S A
SA
McElwain
T F
TF
D000818:Animals; D001403:Babesia / Q000235:genetics* / Q000378:metabolism; D016793:Babesia bovis / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D016384:Consensus Sequence; D017124:Conserved Sequence; D005786:Gene Expression Regulation / Q000235:genetics*; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D015800:Protozoan Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry; D014158:Transcription, Genetic
D015800:Protozoan Proteins; D012333:RNA, Messenger; C097551:rhoptry-associated protein-1, Babesia