Hodgkinson, Colin Andrew; (1990) Studies on the expression and functional activities of bacteriophage T7 RNA polymerase and vaccinia virus guanylyltransferase in mammalian cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The ability to express exogenously introduced genes in mammalian cells under control of strong viral promoters has been valuable in many areas of cell and molecular biology. Transcription from these promoters is driven by the host cell's RNA polymerases, together with an array of host-derived transcription factors, which influence the level and cell-type specificity of transcription in as yet poorly understood ways. Transcription of exogenous genes by a bacteriophage RNA polymerase in mammalian cells could offer several potential advantages: lack of inherent cell-type specificity, high transcription rates, exquisite promoter specificity, and the possibility of utilising well understood and characterised procaryotic gene control mechanisms. Potential problems exist, arising from the different requirements for RNA processing in procaryotic and eucaryotic cells. This thesis describes investigations into the feasibilty of using T7 RNA polymerase to generate functional mRNAs in mammalian cells. Introduction of the bacterial choloramphenicol acetyltransferase (CAT) gene under the transcriptional control of a late T7 promoter into mammalian cells together with a eucaryotic expression vector encoding T7 RNA polymerase, by transfection or microinjection did not result in expression of CAT, suggesting a block to transcription and/or post- transcriptional RNA processing. This block was overcome by superinfection of transfected cells with vaccinia virus, which carries its own RNA processing enzymes, including a guanylyltransferase which modifies free 5'-ends by addition of a guanylyl residue in a 5'-5' linkage. Such capping of eucaryotic mRNAs is ubiquitous and essential for mRNA stability and translation initiation. I cloned the vaccinia virus guanylyltransferase (GT) gene into a eucaryotic expression vector to investigate whether expression of cloned GT could cooperate and complement TV RNA polymerase to generate stable translatable mRNAs in mammalian cells. Expression of the cloned GT was unable to complement T7 RNA polymerase activity, and antiserum generated against a bacterial fusion protein containing the C-terminal third of GT revealed the expressed GT to be located at discrete sites within the nucleus. My results suggest that either further RNA processing events are required to convert T7 transcripts to functional mRNAs, or that the GT Protein/and T7 transcripts are sequestered into different regions of the nucleus and do not encounter each other.

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