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Sequence and functional analysis of the intergenic regions separating babesial rhoptry-associated protein-1 (rap-1) genes.
Title | Sequence and functional analysis of the intergenic regions separating babesial rhoptry-associated protein-1 (rap-1) genes. |
Publication Type | Journal Article |
Year of Publication | 1998 |
Authors | Suarez, CE, Palmer, GH, Hötzel, I, Hines, SA, McElwain, TF |
Journal | Exp Parasitol |
Volume | 90 |
Issue | 2 |
Pagination | 189-94 |
Date Published | 1998 Oct |
ISSN | 0014-4894 |
Keywords | Animals, Babesia, Babesia bovis, Base Sequence, Consensus Sequence, Conserved Sequence, Gene Expression Regulation, Genes, Protozoan, Molecular Sequence Data, Multigene Family, Promoter Regions, Genetic, Protozoan Proteins, RNA, Messenger, Transcription, Genetic |
Abstract | 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. |
DOI | 10.1006/expr.1998.4321 |
Alternate Journal | Exp. Parasitol. |
PubMed ID | 9769249 |