Viral promoters can initiate expression of toxin genes introduced into Escherichia coli.
BACKGROUND
The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures.


RESULTS
We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene.


CONCLUSIONS
According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.
Lewin
Astrid
A
Robert Koch-Institut, Nordufer 20, 13353 Berlin, Germany. Lewina@rki.de
Mayer
Martin
M
Chusainow
Janet
J
Jacob
Daniela
D
Appel
Bernd
B
D001426:Bacterial Proteins / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology / Q000379:methods*; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics; D003603:Cytotoxins / Q000235:genetics; D004247:DNA / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D015497:HIV-1 / Q000235:genetics; D006460:Hemolysin Proteins / Q000378:metabolism; D006461:Hemolysis; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D049449:Luminescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D000077183:Occlusion Body Matrix Proteins; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D013539:Simian virus 40 / Q000235:genetics; D020079:Terminal Repeat Sequences; D013937:Thymidine Kinase / Q000235:genetics / Q000378:metabolism; D014118:Toxins, Biological / Q000378:metabolism*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics; D015678:Viral Structural Proteins
D001426:Bacterial Proteins; D003603:Cytotoxins; D006460:Hemolysin Proteins; D000077183:Occlusion Body Matrix Proteins; D014118:Toxins, Biological; D014764:Viral Proteins; D015678:Viral Structural Proteins; C009829:polyhedrin protein, Nucleopolyhedrovirus; C110221:vllY protein, Vibrio vulnificus; D004247:DNA; D008156:Luciferases; D013937:Thymidine Kinase