Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A dual eukaryotic/prokaryotic expression vector has been developed which combines the features of positive selection for cloned inserts along with the production of an epitope-tagged cDNA insert by transient transfection in mammalian cells as well as high level induced expression in E. coli cells harbouring T7 RNA polymerase. This vector, pZilch, has two MCSs flanking a mutant E. coli phenylalanyl-tRNA synthetase gene, pheS, which when expressed in combination with the phenylalanine analog p-CI-Phe, results in termination of host cell protein synthesis. Cloning of inserts using unique sites in the flanking MCS regions results in loss of the pZilch pheS allele and hence permits growth of colonies harbouring recombinants on p-Cl-Phe plates. Additional features of the vector include an optimal Kozak consensus sequence for high level eukaryotic cell expression and an efficient prokaryotic translation initiation site in frame and downstream from the eukaryotic initiation site. Recombinant proteins can be produced with an N-terminal FLAG epitope which can be removed via a specific protease cleavage site. Flanking T7 and SP6 RNA polymerase promoter sites permit in vitro transcription and translation of cloned inserts. A derivative of the vector has also been constructed enabling nuclear accumulation of the tagged proteins via an SV40 nuclear localisation signal upstream of the 5' MCS.
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PMID:An epitope tagged mammalian/prokaryotic expression vector with positive selection of cloned inserts. 933 83

Based on the highly conserved sequences of small nuclear RNA and small cytoplasmic RNA between vertebrate species, three porcine type III RNA polymerase III promoters, pY1, pY3 and pU6, were identified by using genomic DNA walking. To test the functional relationship of these sequences, the human H1 promoter of pSUPER-EGFP-l-neo vector was substituted with these three promoters to create the ppPol III-MCS vectors. The strength of each promoter was measured by its ability to derive expression of shRNA to repress expression of luciferase via RNA interference in the pig kidney epithelial cell line LLC-PK1. We determine that the ranking of promoter strength in descending order is pU6 > pY1 > pY3.
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PMID:Porcine type III RNA polymerase III promoters for short hairpin RNA expression. 1916 86