当前位置： SCI文献检索 > PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA期刊下所有文献 > Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system.

Cap-independent translation of mRNA conferred by encephalomyocarditis virus 5' sequence improves the performance of the vaccinia virus/bacteriophage T7 hybrid expression system.

Abstract：

:A recombinant vaccinia virus that directs the synthesis of bacteriophage T7 RNA polymerase provides the basis for the expression of genes that are regulated by T7 promoters in mammalian cells. The T7 transcripts, which account for as much as 30% of the total cytoplasmic RNA at 24 hr after infection, are largely uncapped. To improve the translatability of the uncapped RNA, the encephalomyocarditis virus (EMCV) untranslated region (UTR) was inserted between the T7 promoter and the chloramphenicol acetyltransferase (CAT) gene. Experiments with a reticulocyte extract demonstrated that the EMCV UTR conferred efficient and cap-independent translatability to CAT RNA synthesized in vitro by T7 RNA polymerase. In cells infected with recombinant vaccinia viruses containing the T7 promoter-regulated CAT gene, the EMCV UTR increased the amount of CAT RNA on polyribosomes. The polyribosome-derived CAT RNA, which contained the EMCV UTR, was translated in vitro in a cap-independent fashion as well. Use of the EMCV UTR significantly enhanced the vaccinia/T7 hybrid expression system as it resulted in a 4- to 7-fold increase in total CAT activity. A further approximately 2-fold improvement was achieved by incubating the cells in hypertonic medium, which favors the translation of uncapped picornavirus RNA over cellular mRNAs. With this newly modified expression system, CAT was the predominant protein synthesized by infected cells and within 24 hr accounted for greater than 10% of the total cell protein.

journal_name

Proc Natl Acad Sci U S A

journal_title

Proceedings of the National Academy of Sciences of the United States of America

authors

Elroy-Stein O,Fuerst TR,Moss B

doi

10.1073/pnas.86.16.6126

subject

Has Abstract

pub_date

1989-08-01 00:00:00

pages

6126-30

issue

16

eissn

0027-8424

issn

1091-6490

journal_volume

86

pub_type

杂志文章

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