The eight negative-strand RNA genome segments of fowl plague virus, an influenza A virus, were polyadenylated in vitro at their 3' termini using an enzyme, poly(A)polymerase, which was isolated from E. coli. The subsequent use of oligo(dT) to prime reverse transcription of this RNA, and of mRNA isolated from fowl plague virus-infected chick embryo fibroblasts (which contains poly(A)), allowed the production of full-length DNA copies of both classes of influenza virus RNA. These single-strand cDNAs were annealed together to produce individual dsDNAs corresponding to each segment of the influenza virus genome. dsDNAs produced by this approach were used for molecular cloning in plasmid pBR322, and a recombinant plasmid containing influenza virus-specific sequences derived from genome segment 7 (the matrix gene) was isolated. Direct sequence analysis of this plasmid demonstrated the presence of additional, non-virus coded sequences at the 5' end of matrix gene mRNA. A specific restriction fragment derived from this plasmid was used for sequence analysis of the 5' region of a population of matrix gene mRNAs by the dideoxy-chain termination method of DNA sequencing, demonstrating that the additional host-derived region is heterogeneous in both sequence and length, and covers a size range of approximately 9-15 nucleotides. In addition, these analyses indicated that during transcription of matrix gene mRNA, the 3' terminal nucleotide present in virion RNA is not transcribed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:597369 |
| Date | January 1981 |
| Creators | Caton, A. J. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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