TY - JOUR
T1 - The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo
AU - Gallie, Daniel R.
AU - Sleat, David E.
AU - Watts, John W.
AU - Turner, Philip C.
AU - Wilson, T. michael A.
N1 - Funding Information:
Professors Milton Zaitlin and Harold Woolhouse, and Dr. Roger Hull, for constructive comments on the text. This work was funded by Diatech Ltd., London and in part by a grant-in-aid from the Agricultural and Food Research Council to the John Innes Institute. D.E.S. was supported by a CASE studentship from the Science and Engineering Research Council. These results form part of New British Patent Application No. 86 13481.
PY - 1987/4/24
Y1 - 1987/4/24
N2 - A 67-nucleotide portion of the non-coding, 5′-leader sequence of tobacco mosaic virus RNA [defined as Ω' (Gr. omega prime)] has been shown to enhance the translation of contiguous foreign gene transcripts both in vitro and in vivo. Chemically-synthesized Ω', containing convenient linker sequences, was inserted into derivatives of an in vitro transcription plasmid (pSP64) between the bacteriophage-SP6 promoter and sequences coding for either chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII). Run-off in vitro transcripts, with or without a 5′-cap structure (G(5′)ppp(5′)G) and/or the Ω' sequence, were tested in mRNA-dependent cell-free translation systems derived from rabbit reticulocyte lysate, wheat germ extract or Escherichia coli (MRE 600). In all cases, the presence of Ω' increased the translational expression of both reporter genes, typically between 2- to 10-fold. Electroporation of isolated mesophyll protoplasts from Nicotiana tabacum cv. Xanthi, or microinjection of oocytes from Xenopus laevis, with SP6-transcripts containing the CAT-coding region confirmed and extended the value of Ω' as a potential translational enhancer of gene expression in vivo.
AB - A 67-nucleotide portion of the non-coding, 5′-leader sequence of tobacco mosaic virus RNA [defined as Ω' (Gr. omega prime)] has been shown to enhance the translation of contiguous foreign gene transcripts both in vitro and in vivo. Chemically-synthesized Ω', containing convenient linker sequences, was inserted into derivatives of an in vitro transcription plasmid (pSP64) between the bacteriophage-SP6 promoter and sequences coding for either chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII). Run-off in vitro transcripts, with or without a 5′-cap structure (G(5′)ppp(5′)G) and/or the Ω' sequence, were tested in mRNA-dependent cell-free translation systems derived from rabbit reticulocyte lysate, wheat germ extract or Escherichia coli (MRE 600). In all cases, the presence of Ω' increased the translational expression of both reporter genes, typically between 2- to 10-fold. Electroporation of isolated mesophyll protoplasts from Nicotiana tabacum cv. Xanthi, or microinjection of oocytes from Xenopus laevis, with SP6-transcripts containing the CAT-coding region confirmed and extended the value of Ω' as a potential translational enhancer of gene expression in vivo.
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U2 - 10.1093/nar/15.8.3257
DO - 10.1093/nar/15.8.3257
M3 - Article
C2 - 3575095
AN - SCOPUS:0023663363
SN - 0305-1048
VL - 15
SP - 3257
EP - 3273
JO - Nucleic acids research
JF - Nucleic acids research
IS - 8
ER -