Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/1/2006
Publication Date: 7/5/2006
Citation: Suarez, C.E., Norimine, J., Lacy, P.A., Mcelwain, T.F. 2006. Characterization and gene expression of Babesia bovis elongation factor-1alpha. International Journal for Parasitology. 36(8):965-973. Interpretive Summary: Babesia bovis is a causative agent of bovine babesiosis. In this study, we identified and characterized the two genes encoding for the elongation factor-1 alpha of Babesia bovis and we utilized a transient transfection system to characterize the ef-1alpha promoter located in the intergenic region (IG) separating both genes. This study demonstrates that the ef-1alpha IG region of B. bovis contains strong promoters able to express both ef-1alpha genes, and can efficiently promote expression of foreign genes in transiently transfected parasites. Since the development of robust transient and stable transfection systems can be significantly enhanced by high level expression of exogenous genes, use of ef-1alpha regulatory regions may be beneficial in developing stable transformation systems for B. bovis parasites.
Technical Abstract: Elongation factor 1 alpha (EF-1') is a constitutively expressed, abundant protein that is a key element in eukaryotic protein translation. Because of its high level of transcription, the EF-1''promoter has been utilized to drive exogenous gene expression in transfected cells. In this study, we identified and characterized the ef-1' locus of Babesia bovis, a causative agent of bovine babesiosis, and examined the transcriptional activity of the EF-1' promoter' The ef-1' locus in the T2Bo strain of Babesia bovis contains two identical ef-1' genes (“A” and “B”) arranged in a head to head orientation and separated by a 1.4 kb intergenic (IG) region containing a 260 bp terminal inverted repeat. Both ef-1' genes encode identical proteins with 448 amino acids and a calculated molecular mass of 49 kD. While the B. bovis ef-1' -IG sequence is conserved among multiple strains of B. bovis, it is not significantly related to any regulatory sequence in the DNA databases. The IG region promotes expression of both ef-1' genes. Both, fragment Ig-A containing 730 bp upstream of ef-1' open reading frame A, and fragment Ig-B, containing 720 bp upstream of ef-1' open reading frame B, were able to promote luciferase in transient transfection. In the 5’'3’ orientation, the Ig-B fragment resulted in the highest level of luciferase activity, 10 times higher than positive control plasmid p40-15-luc containing the rap-1 IG region, suggesting that this fragment contains a very strong promoter. Analysis of ef-1' transcripts confirms that both ef-1''genes are transcribed in merozoites. Interestingly, in contrast to other related intra-erythrocytic apicomplexans, the ef-1' locus of B. bovis contains a 160 bp intron in the 5’ utr region.