Oriented synthesis and cloning of influenza virus nucleoprotein cDNA that leads to its expression in mammalian cells.
The influenza virus nucleoprotein gene has been cloned by a procedure that involves direct cDNA synthesis onto the primer-vector pBSV9, a pBR322-SV40 recombinant plasmid. dT-tailed pBSV9 was used to prime the synthesis of cDNA on a template of in vitro synthesized viral mRNA. The synthesis of ds-cDNA was initiated by a specific oligodeoxynucleotide and the resulting recombinant was circularized by intramolecular ligation. Recombinant pSVa963 contained the viral nucleoprotein gene directly fused to the SV40 early promoter region included in pBSV9 and followed by a dA:dT tail and the SV40 polyadenylation signal. When pSVa963 was used to transfect COS-1 cells, the presence of three NP-specific mRNAs of 1600, 1900 and 2500 nucleotides in length could be detected. Pulse labelling experiments of COS-1 transfected cells and immunobinding to a nucleoprotein monoclonal antibody indicated the synthesis of nucleoprotein. This nucleoprotein accumulated in the nucleus of transfected cells at a level similar to that found in infected cells. The vector and method described may be useful for the specific cloning and expression of any mRNA for which a 5'-terminal sequence is known.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004285:Dogs; D005796:Genes; D005814:Genes, Viral; D005822:Genetic Vectors; D009980:Influenza A virus / Q000235:genetics*; D007668:Kidney; D009698:Nucleoproteins / Q000235:genetics*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic
D009698:Nucleoproteins; D012333:RNA, Messenger; D004247:DNA; D004262:DNA Restriction Enzymes