Efficient expression of a protein coding gene under the control of an RNA polymerase I promoter.
In mammalian cells, RNA polymerase I transcripts are uncapped and retain a polyphosphate 5' terminus. It is probably for this reason that they are poorly translated as messenger RNA. We show in this report that insertion of an Internal Ribosome Entry Site (IRES) into the 5' leader of an RNA polymerase I transcript overcomes the block to translation, presumably by substituting for the 5' trimethyl G cap. Addition of an SV40 polyA addition signal also enhances protein production from the RNA polymerase I transcript. RNA Polymerase I driven expression vectors containing both elements produce protein at levels comparable to that produced from RNA polymerase II driven expression vectors which utilize a retroviral LTR. RNA Polymerase I driven expression vectors may have a variety of uses both for basic research and for practical expression of recombinant proteins.
Hutchinson Cancer Research Center, Seattle, WA 98104.
T D
TD
Palmer
A D
AD
Miller
R H
RH
Reeder
B
B
McStay
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004275:DNA, Ribosomal / Q000235:genetics; D015246:Deoxyribonuclease EcoRI; D004351:Drug Resistance / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D019868:Kanamycin Kinase; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009355:Neomycin; D010770:Phosphotransferases / Q000235:genetics; D010957:Plasmids; D011061:Poly A / Q000235:genetics; D011401:Promoter Regions, Genetic; D012318:RNA Polymerase I / Q000378:metabolism*; D051381:Rats; D013539:Simian virus 40 / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D004275:DNA, Ribosomal; D011061:Poly A; D010770:Phosphotransferases; D019868:Kanamycin Kinase; D012318:RNA Polymerase I; D015246:Deoxyribonuclease EcoRI; D009355:Neomycin