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