title: Structure, sequence, and transcriptional analysis of the Babesia bovis rap-1 multigene locus.
abstract: The complexity of multigene families encoding rhoptry proteins and the generation of new variants in these families are constraints to development of vaccines incorporating rhoptry proteins. For example, the Babesia bigemina rhoptry associated protein (rap)-1 locus is composed of tandemly arranged genes including four polymorphic rap-1a genes and two classes of divergent genes, rap-1b and rap-1c. B. bigemina rap-1 polymorphism reflects recombination and gene conversion and results in multiple RAP-1 proteins with unique B- and T-cell epitopes. Is this complex locus structure and recombination a required feature of the rap-1 gene family among Babesia species? We addressed this question by analysis of the rap-1 locus in B. bovis. Sequence analysis of an 11 kb genomic clone representing the B. burn rap-1 locus revealed only two identical and continuous rap-1a gene copies, rap 1a-1 and rap-1a-2, located in a similar head to tail orientation. Using the conserved ig gene as a marker for the 3' boundary of the rap-1 locus, we conclude that divergent rap-1b and rap-1c genes, present in B. bigemina, are not similarly cis-linked to the B. bovis rap-1 locus. Analysis of the rap-1a genes 1 and 2 from each of multiple B. bovis strains from North and South America demonstrated RAP-1 size conservation with very limited amino acid sequence variation. The results suggest that the simple two gene arrangement in the B. bovis rap-1 gene family was generated by gene duplication and, in contrast to the B. bigemina rap-1 locus, both genes evolved together using homogenization mechanisms with point mutation as the single mechanism for gene variation. Three discontinuous non-rap-1 genes are closely cis-linked to the B. bovis rap-1 locus and the presence of multiple introns in these genes may limit rap-1 gene variation due to unequal crossing over. The different mechanisms likely involved in the evolution of the rap-1 family in B. bigemina versus B. bovis are reflected in the marked structural and antigenic polymorphism in the B. bigemina RAP-1 molecules as compared with the essentially monomorphic RAP-1 in B. bovis.
authors.affiliation: Department of Veterinary Microbiology and Pathology, Washington State University, Pullman 99164-7040, USA. ces@vetmed.wsu.edu
authors.forename: C E
authors.initials: CE
authors.lastname: Suarez
authors.forename: G H
authors.initials: GH
authors.lastname: Palmer
authors.forename: I
authors.initials: I
authors.lastname: Hötzel
authors.forename: T F
authors.initials: TF
authors.lastname: McElwain
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000737:chemistry / Q000235:genetics; D016793:Babesia bovis / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D017125:Genes, Protozoan; D014644:Genetic Variation; D007438:Introns; D008969:Molecular Sequence Data; D005810:Multigene Family; D016366:Open Reading Frames; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
chemical_list: D000953:Antigens, Protozoan; D015800:Protozoan Proteins; C097551:rhoptry-associated protein-1, Babesia