Full-length-enriched cDNA libraries from Echinococcus granulosus contain separate populations of oligo-capped and trans-spliced transcripts and a high level of predicted signal peptide sequences

摘要

The tissue-dwelling larval stages of the cestode udEchinococcus granulosus are intimately associated with the host, implying that audrange of molecular mediators may be secreted by the parasite into the host environment. These mediators are being sought throughuda transcriptome-based analysis, using recombinant cDNA libraries. Conventional cDNA libraries of E. granulosus contain highudlevels of mitochondrial transcripts, as well as host (bovine) genomic DNA . In particular, 60% of a conventional protoscolex stageudcDNA library corresponds to the large subunit (L SU) of mitochondrial rRNA . We attribute the presence of LSU rRNA copies to itsudpolyadenylation in E.granulosus. To circumvent this problem, we adapted the 5ı Rapid Amplification of cDNA Ends (RNA -ligaseudmediated RACE) technique that excludes all polynucleotides missing the 7-methyl-guanosine (7MG) cap specific to the 5ı end ofudfull-length mRNA. By ligating a specific oligonucleotide (oligo-cap) to 7MG -bearing mRNA , three cDNA libraries were made byudPCR from oligo-cap and oligo-dT primers. Analysis of these libraries showed that mitochondrial RNA contaminants had beenudexcluded. Moreover, no bovine genomic sequences were detected. In parallel, we constructed three cDNA libraries using the newlyuddescribed trans-spliced leader (SL) from Echinococcus. Although these represent a smaller subset of parasite genes, mitochondrialudand genomic contributions were again excluded. In both cases, a majority of cDNA s (61ˇ/92%) were judged to contain the initiationudA T G codon, and 11ˇ /27% of inserts included potential N-terminal signal sequences. The 5ı UTR tracts of most oligo-capped cDNAsudwere ı /100 nt, although ˇ /8% were longer than this. Among the trans-spliced cDNA s, 43% potentially utilise the AU G donated byudthe SL , and in only 6% was the SL separated from an endogenous putative start site by 60 nt. Sequence analysis of randomlyudselected clones shows virtually no overlap between the oligo-capped and SL libraries, indicating that trans-spliced E.granulosusudmR NA s appear to be insensitive to the enzymatic treatments used to ‘oligo-cap’ unspliced mRNAs. The oligo-capped and SLudstrategies represent efficient and complementary pathways to isolate full-length cDNA clones from this cestode parasite and,udpossibly, from related parasitic flatworms.