A trimmed viral cap-independent translation enhancing sequence for rapid in vitro gene expression.
We prepared a short (29 nucleotides) 5' UTR that enhanced cap-independent translation in a wheat germ translation system by trimming the tobacco etch virus 5' UTR. The trimmed sequence, designated as TE(37-65), was obtained from a conserved region among several potyviruses. The productivities of uncapped reporter mRNAs carrying the TE(37-65) sequence were comparable to those of capped counterparts, in that 5-20 microg of proteins were synthesized per 1 mL of translation reaction mixture. The ribosome that entered onto the TE(37-65) sequence precisely initiated polypeptide synthesis at the defined initiation codon, which ensures rapid and efficient protein truncation analyses. Moreover, the TE(37-65) sequence is short enough to be involved in a PCR primer, which allows a simple method for rapid gene expression, i.e., PCR amplification of a target gene and succeeding in vitro transcription and translation. As a demonstration, the rapid in vitro expression of rice cDNAs using the TE(37-65) sequence was also performed.
Kawarasaki
Y
Y
Laboratory of Molecular Biotechnology, Division of Molecular and Cell Mechanisms, Department of Biological Mechanisms and Functions, Graduate School of Bio- and Agro-Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Kasahara
S
S
Kodera
N
N
Shinbata
T
T
Sekiguchi
S
S
Nakano
H
H
Yamane
T
T
D020121:5' Untranslated Regions; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D015870:Gene Expression; D010942:Plant Viruses / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis / Q000235:genetics*; D012315:RNA Caps; D017386:Sequence Homology, Amino Acid
D020121:5' Untranslated Regions; D004279:DNA, Viral; D012315:RNA Caps