Utilisation de systèmes de vecteurs plasmidiques aux fins d'identification de promoteurs géniques.
Creators: name: Leclair, Benoît.
Publisher: name: Université d'Ottawa / University of Ottawa
Year: 1990
Subjects: subject: Biology, Genetics.

description: The understanding of the mechanisms involved in the control of gene expression is an important step towards understanding the nature of cellular events such as cellular determination and differentiation. We have studied one of many mechanisms by which the control of gene expression is exercised, namely the control of the rate of initiation of transcription. We have developed a plasmid-based system for the search and characterization of transcriptional promoters and enhancers within small size genomes. The system was designed to select for strong promoting and enhancing elements capable of substitution for those of the early region of SV40. It allows for the use of different reporter genes and various measurement protocols. We have used part of the viral genome of herpes simplex type 2 to test our system. We have searched a transforming fragment of HSV-2 for the presence of transcriptional enhancers and examined the transcriptional activity on the fragment. We have found no transcriptional enhancer within the fragment, suggesting that transformation is not accomplished through the action of a viral enhancer. The results suggest, as far as carcinogenesis is concerned, that a viral $\beta$-class gene would be a likely target for transcriptional enhancement by a cellular element, should an integration event place the transforming fragment near one. The use of SV40's large T antigen as the reporter gene has allowed us to observe, in rare HeLa cells, normal level production of large T when transcription of the reporter gene is "enhanced" by the presence within the vector of certain sub-fragments of the transforming fragment. Three sub-fragments found in the cellular genome of HSV-2-transformed cells have generated a similar result. We have also shown that within clones of HeLa cells transfected with these herpes-derived constructs, the promoter of one or two integrated copies transcribe at the same level as the promoter and enhancer of SV40. One interpretation of these results suggests that the three sub-fragments somehow direct the integration of the plasmids to transcriptionally active chromosomal locations. We have used the vector system to further characterize an enhancer found upstream of the mouse phosphoglycerate kinase-1 gene (pgk-1). This enhancer induces a 30-fold increase in transcription activity from the pgk-1 promoter in mouse cells. When linked to the SV40 early promoter, the transcriptional enhancement is only marginal. We concluded that the mouse pgk-1 enhancer, unlike the SV40 enhancer, is unable to enhance heterologous promoters. We have used an expression vector where SV40's large T antigen is under the transcriptional control of the mouse pgk-1 promoter to assess whether or not the presence of large T can alter the normal progression of differentiation of the teratocarcinoma cell line P19. We have generated large T-expressing P19 clones. The differentiation of these clones as a monolayer has produced homogeneous populations of differentiated progeny, the common phenotype varying from one clone to the other. One interpretation of these results suggests that cellular determination had already been achieved within these clones. (Abstract shortened by UMI.)