Linked Life Data

12106871

Source:http://linkedlifedata.com/resource/pubmed/id/12106871

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2002-7-10
pubmed:abstractText
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 (LSU) of mitochondrial rRNA. We attribute the presence of LSU rRNA copies to its polyadenylation in E. granulosus. To circumvent this problem, we adapted the 5' Rapid Amplification of cDNA Ends (RNA-ligase mediated RACE) technique that excludes all polynucleotides missing the 7-methyl-guanosine (7MG) cap specific to the 5' 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 cDNAs (61-92%) were judged to contain the initiation ATG codon, and 11-27% of inserts included potential N-terminal signal sequences. The 5' UTR tracts of most oligo-capped cDNAs were <100 nt, although approximately 8% were longer than this. Among the trans-spliced cDNAs, 43% potentially utilise the AUG 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 mRNAs 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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jul
pubmed:issn
0166-6851
pubmed:author
pubmed:issnType
Print
pubmed:volume
122
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
171-80
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:12106871-5' Untranslated Regions, pubmed-meshheading:12106871-Animals, pubmed-meshheading:12106871-Base Sequence, pubmed-meshheading:12106871-Cattle, pubmed-meshheading:12106871-DNA, Complementary, pubmed-meshheading:12106871-Databases, Factual, pubmed-meshheading:12106871-Echinococcus, pubmed-meshheading:12106871-Expressed Sequence Tags, pubmed-meshheading:12106871-Gene Library, pubmed-meshheading:12106871-Molecular Sequence Data, pubmed-meshheading:12106871-Oligonucleotides, pubmed-meshheading:12106871-Open Reading Frames, pubmed-meshheading:12106871-Protein Sorting Signals, pubmed-meshheading:12106871-RNA, pubmed-meshheading:12106871-RNA, Helminth, pubmed-meshheading:12106871-RNA, Messenger, pubmed-meshheading:12106871-RNA, Spliced Leader, pubmed-meshheading:12106871-RNA Caps, pubmed-meshheading:12106871-Transcription, Genetic
pubmed:year
2002
pubmed:articleTitle
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.
pubmed:affiliation
Institute of Cell, Animal and Population Biology, University of Edinburgh, West Mains Road, UK.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't, Evaluation Studies