Journal article
Intergenic regions in the rhoptry associated protein-1 ( rap-1) locus promote exogenous gene expression in Babesia bovis
International journal for parasitology, Vol.34(10), pp.1177-1184
2004
Handle:
https://hdl.handle.net/2376/107715
PMID: 15380689
Abstract
Members of the
Babesia
rap-1 gene family are expressed during multiple parasite stages, and are regulated by both transcriptional and post-transcriptional mechanisms. In all
Babesia species, tandemly arranged
rap-1 gene copies are separated by an intergenic (IG) region that is hypothesized to regulate gene expression. In this study, we tested that hypothesis by determining whether the
Babesia bovis
rap-1 IG region could promote extra-chromosomal expression of exogenous genes introduced into merozoites by transfection, and whether a tandem arrangement of IG regions similar to the
rap-1 locus enhances exogenous gene expression. Initially, electroporation conditions of
B. bovis parasites were determined using expression of the reporter luciferase gene. Both
B. bovis transfected by electroporation and
Escherichia coli transformed with plasmid
p40-15-luc containing the luciferase gene under the control of the
B. bovis
rap-1 IG and 3′ flanking regions were able to express luciferase, indicating that the
rap-1 IG region contains a functional promoter. The chromosomal organization of the
B. bovis
rap-1 locus includes two identical
rap-1 open reading frames and IG regions in a head to tail orientation. To determine whether this orientation enhanced expression of exogenous genes, plasmid constructs containing two
rap-1-IG regions controlling expression of the
luc and human dihydrofolate reductase (
hdhfr) genes, and oriented either in head to head (
pLuc-H-13) or head to tail (
pLuc-H-18) arrangement, were compared. The head to tail orientation of the gene cassettes resulted in a significant increase in the level of luciferase as compared to either head to head orientation or a single IG region construct (
p40-15-luc). Thus, an organization that mimics the native structure of the
rap-1 locus results in enhanced luciferase expression. These results are the first to demonstrate exogenous gene expression in
B. bovis after transfection, and to confirm that the
B. bovis
rap-1 IG region can promote extra-chromosomal gene expression in vivo.
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Details
- Title
- Intergenic regions in the rhoptry associated protein-1 ( rap-1) locus promote exogenous gene expression in Babesia bovis
- Creators
- Carlos E Suarez - Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USAGuy H Palmer - Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USATanya LeRoith - Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USAMonica Florin-Christensen - Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USABrendan Crabb - The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, 3050 Melbourne, AustraliaTerry F McElwain - Program in Vector-Borne Diseases, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164-7040, USA
- Publication Details
- International journal for parasitology, Vol.34(10), pp.1177-1184
- Academic Unit
- Veterinary Microbiology and Pathology, Department of; Paul G. Allen School for Global Animal Health
- Publisher
- Elsevier Ltd
- Identifiers
- 99900547239701842
- Language
- English
- Resource Type
- Journal article