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In vitro synthesis of full‐length influenza virus complementary RNA.
Author(s) -
Río L.,
Martínez C.,
Domingo E.,
Ortín J.
Publication year - 1985
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1985.tb02342.x
Subject(s) - biology , rna , in vitro , polyadenylation , virus , microbiology and biotechnology , virology , gene , genetics
Influenza virus‐specific RNA has been synthesized in vitro, using cytoplasmic or microsomal fractions of influenza virus‐infected MDCK cells. The RNA polymerase activity was stimulated 5‐30 times by priming with ApG. About 20‐30% of the product was polyadenylated. Most of the in vitro product was of positive polarity, as shown by hybridization to strand specific probes and by T1 fingerprinting of the poly(A)+ and poly(A)‐ RNA segments encoding haemagglutinin and nucleoprotein. The size of poly(A)‐ RNA segments, determined on sequencing gels, was indistinguishable from that of virion RNA, whereas poly(A)+ RNA segments contain poly(A) tails approximately 50 nucleotides long. The size of in vitro synthesized RNA segments was also determined by gel electrophoresis of S1‐treated double‐stranded RNAs, obtained by hybridization of poly(A)+ or poly(A)‐ RNA fractions with excess of unlabelled virion RNA. The results of these experiments indicate that poly(A)‐ RNA contains full‐length complementary RNA. This conclusion is further substantiated by the presence of additional oligonucleotides in the T1 fingerprints of in vitro synthesized poly(A)‐ haemagglutinin or nucleoprotein RNA, selected by hybridization to cloned DNA probes corresponding to the 3′ termini of the genes.