TY - JOUR
T1 - Expression of Toxoplasma gondii genes in the closely-related apicomplexan parasite Neospora caninum
AU - Howe, Daniel K.
AU - Mercier, Corinne
AU - Messina, Marinella
AU - Sibley, L. David
N1 - Funding Information:
We thank J. Boothroyd (Stanford University) for providing the pSAG1- β -Gal and the P30.5COS1.1.6 plasmids, M.F. Cesbron (Pasteur Institute, Lille) for the G43 plasmid and the Tg17-179 antibody, and D. Lindsay (Auburn University) for the Nc-1 strain of N. caninum . DKH was partially supported by a NIH training grant (AI07172). LDS is a Burroughs Wellcome Funded New Investigator in Molecular Parasitology.
PY - 1997/5
Y1 - 1997/5
N2 - We have established the feasibility of using Neospora caninum as a heterologous system for the expression of genes from the closely-related parasite Toxoplasma gondii. Plasmid constructs containing the lacZ gene from Escherichia coli driven by T. gondii promoters were electroporated into N. caninum parasites, and expression of β-galactosidase (β-Gal) activity was assayed enzymatically. In transient assays, expression of β-Gal driven by the GRA1 promoter was approximately 10-fold higher than the expression obtained with the SAG1 promoter. Enzyme activity was not detected when N. caninum parasites were transfected with a promoterless lacZ construct. Transfection of N. caninum with complete genomic clones of SAG1 or GRA2 from T. gondii yielded parasites that transiently expressed the respective gene products, as detected by immunofluorescence and Western blot. Additionally, these transiently expressed T. gondii proteins appeared by immunofluorescence localization and Triton X-114 partitioning to be correctly targeted in both extracellular and intracellular N. caninum parasites. Heterologous gene expression should be useful for studying the function of specific gene products and may facilitate the identification of genes responsible for the phenotypic differences observed between these two closely-related apicomplexan parasites.
AB - We have established the feasibility of using Neospora caninum as a heterologous system for the expression of genes from the closely-related parasite Toxoplasma gondii. Plasmid constructs containing the lacZ gene from Escherichia coli driven by T. gondii promoters were electroporated into N. caninum parasites, and expression of β-galactosidase (β-Gal) activity was assayed enzymatically. In transient assays, expression of β-Gal driven by the GRA1 promoter was approximately 10-fold higher than the expression obtained with the SAG1 promoter. Enzyme activity was not detected when N. caninum parasites were transfected with a promoterless lacZ construct. Transfection of N. caninum with complete genomic clones of SAG1 or GRA2 from T. gondii yielded parasites that transiently expressed the respective gene products, as detected by immunofluorescence and Western blot. Additionally, these transiently expressed T. gondii proteins appeared by immunofluorescence localization and Triton X-114 partitioning to be correctly targeted in both extracellular and intracellular N. caninum parasites. Heterologous gene expression should be useful for studying the function of specific gene products and may facilitate the identification of genes responsible for the phenotypic differences observed between these two closely-related apicomplexan parasites.
UR - http://www.scopus.com/inward/record.url?scp=0030946239&partnerID=8YFLogxK
U2 - 10.1016/S0166-6851(97)90003-7
DO - 10.1016/S0166-6851(97)90003-7
M3 - Article
C2 - 9178265
AN - SCOPUS:0030946239
SN - 0166-6851
VL - 86
SP - 29
EP - 36
JO - Molecular and Biochemical Parasitology
JF - Molecular and Biochemical Parasitology
IS - 1
ER -