A possible approach to enrich cDNA yields with full-length molecules.
Newcastle disease virus (NDV)-specific hemagglutinin-neuraminidase (HN) mRNAs were used as the model templates for cDNA synthesis. Polyadenylated RNAs were isolated from the particulate fraction of cytoplasmic extracts of NDV-infected cells rather than from nonfractionated extracts. The approach is based on earlier findings that eucaryotic mRNAs are present in cytoplasmic extracts in the form of ribonucleoproteins (mRNPs) rather than as free nucleic acids. The idea of the approach was to separate mRNPs from cell sap RNases prior to RNA extraction in order to minimize partial enzymatic hydrolysis of mRNAs. The presence of the 5' terminus (mRNA sense) in cDNAs synthesized was considered as an indication for the suitability of mRNA templates for cDNA synthesis. The cDNAs were synthesized and cloned in lambda gt10 phage. About 300 phages carrying the HN-specific inserts have been identified among 50,000 recombinants, and nine of them were analyzed for the presence of the HN 5' terminus. It was found that the termini are present in all the clones analyzed. The result is in an agreement with the expectation that removal of cell sap prior to RNA extraction significantly increases the suitability of RNA templates for cDNA synthesis.
Zaslavsky
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Kimron Veterinary Institute, Bet Dagan, Israel.
Molad
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T
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002645:Chickens; D003593:Cytoplasm / Q000032:analysis; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism*; D015707:HN Protein / Q000096:biosynthesis / Q000235:genetics; D009521:Newcastle Disease / Q000235:genetics; D009522:Newcastle disease virus / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000302:isolation & purification; D012261:Ribonucleoproteins / Q000302:isolation & purification
D004279:DNA, Viral; D015707:HN Protein; D012333:RNA, Messenger; D012261:Ribonucleoproteins