Gene structure of the extracellular glutathione S-transferase from Onchocerca volvulus and its overexpression and promoter analysis in transgenic Caenorhabditis elegans.
Two highly similar genes encoding unique extracellular, glycosylated glutathione S-transferases (GSTs) of the human-pathogenic nematode, Onchocerca volvulus (Ov-GST1a and Ov-GST1b), have been isolated and characterised. The genes are approximately 3 kb in length and consist of seven exons interrupted by introns of approximately 100 bp in length, with the exception of intron II, which is approximately 1.6 kb in length. Interestingly, exon I and II encode a signal peptide and an N-terminal extension before sequence homology to other GSTs begins. The 5' flanking region was sequenced and analysed for transcription factor binding sites. Consistent with the lack of a TATA box, analysis of the mRNAs by primer extension showed multiple transcription start sites spread over a 60 bp region. To examine the activity and specificity of the Ov-GST1a gene promoter, we have exploited Caenorhabditis elegans as a heterologous transformation system. To analyse whether transgenic C. elegans are able to carry out processing and post-transcriptional modifications of the Ov-GST1a correctly, the protein was ectopically overexpressed in C. elegans. The parasite-derived Ov-GST1a gene product was correctly processed in transgenic C. elegans and posttranslational modifications, such as signal peptide cleavage and N-glycosylation, were performed successfully. This further demonstrates the potential of C. elegans as a host for expression of candidate vaccine antigens from O. volvulus and affirms the role of C. elegans as a model for parasitic nematodes.
Krause
S
S
Department of Biochemical Parasitology, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Str. 74, 20359 Hamburg, Germany.
Sommer
A
A
Fischer
P
P
Brophy
P M
PM
Walter
R D
RD
Liebau
E
E
D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics*; D017931:DNA Primers; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D017181:Onchocerca volvulus / Q000201:enzymology* / Q000235:genetics; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D017931:DNA Primers; D005982:Glutathione Transferase