A comparison of eukaryotic viral 5'-leader sequences as enhancers of mRNA expression in vivo

The 5'-untranslated leader sequences of several plant RNA viruses, and a portion of the 5'-leader of an animal retrovirus, were tested for their ability to enhance expression of contiguous open reading frames for chloramphenicol acetyltransferase (CAT) or beta-glucuronidase (GUS) in tobacc...

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Bibliographic Details
Published in:Nucleic acids research 1987-11, Vol.15 (21), p.8693-8711
Main Authors: GALLIE, D. R, SLEAT, D. E, WATTS, J. W, TURNER, P. C, WILSON, T. M. A
Format: Article
Language:English
Subjects:
Animals
Avian Sarcoma Viruses - genetics
Base Sequence
Biological and medical sciences
Enhancer Elements, Genetic
Escherichia coli
Escherichia coli - metabolism
Female
Fundamental and applied biological sciences. Psychology
Genetic Vectors
Molecular and cellular biology
Molecular genetics
Molecular Sequence Data
Mosaic Viruses - genetics
Nicotiana - metabolism
Oocytes - metabolism
Plants, Toxic
Protein Biosynthesis
Protoplasts - metabolism
Recombinant Fusion Proteins - biosynthesis
RNA Caps
RNA, Messenger - biosynthesis
Rous sarcoma virus
Translation. Translation factors. Protein processing
Xenopus laevis
Xenopus laevis - metabolism
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Summary:The 5'-untranslated leader sequences of several plant RNA viruses, and a portion of the 5'-leader of an animal retrovirus, were tested for their ability to enhance expression of contiguous open reading frames for chloramphenicol acetyltransferase (CAT) or beta-glucuronidase (GUS) in tobacco mesophyll protoplasts, Escherichia coli and oocytes of Xenopus laevis. Translation of capped or uncapped transcripts was substantially enhanced in almost all systems by the leader sequence of either the U1 or SPS strain of TMV. All leader sequences, except that of TYMV, stimulated expression of 5'-capped GUS mRNA with the native prokaryotic initiation codon context, in electroporated protoplasts. Only the TMV leaders enhanced translation of uncapped GUS mRNAs in protoplasts and increased expression of uncapped CAT mRNA in microinjected X. laevis oocytes. In oocytes, the TYMV leader sequence was inhibitory. In transformed E. coli, the TMV-U1 leader enhanced expression of both the native and eukaryotic context forms of GUS mRNA about 7.5-fold, despite the absence of a Shine-Dalgarno region in any of the transcripts. The absolute levels of GUS activity were all about 6-fold higher with mRNAs containing the native initiation codon context. In E. coli, the leaders of AlMV RNA4 and TYMV were moderately stimulatory whereas those of BMV RNA3, RSV and the SPS strain of TMV enhanced GUS expression by only 2- to 3-fold.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/15.21.8693