CaMV gene expression: The analysis of two CaMV promoters in yeast and higher plants.
thesisposted on 19.11.2015, 08:53 by Jennifer H. Richardson
The aim of this study was to assess the feasibility of using the budding yeast Saccharomyces cerevisiae as a system in which to analyse plant promoters. The promoters chosen for study were the 19S and 35S promoters of cauliflower mosaic virus (CaMV) which, like cellular plant promoters, are transcribed in the plant nucleus by host cell RNA polymerase II. A complete CaMV genome was introduced into yeast on a 2 micron plasmid-based vector and using Northern blot analysis, several CaMV-hybridising transcripts were detected. More precise information on the activity of the promoters was obtained by constructing gene fusions in which the 19S and 35S promoters were linked to the bacterial lacZ gene. Biochemical assays for °-galactosidase showed that cells harbouring the 19S-lacZ gene expressed °-galactosidase but those harbouring the 35S-lacZ gene did not. The insertion of a yeast transcription termination signal upstream of the 19S promoter did not abolish or diminish expression of the 19S-lacZ gene. °-galactosidase was present at low levels in cells expressing 19S-lacZ, constituting less than 0.01% of total cell protein. The 5'ends of 19S-lacZ transcripts present in yeast were mapped by primer extension. The major RNA species initiated approximately 250bp upstream of the 19S-lacZ coding region, indicating the existence of a fortuitous promoter in this region of the CaMV DNA. Two less abundant RNA species initiated within the 19S-lacZ open reading frame at positions +9 and +25bp and may be produced from the genuine 19S promoter. There is evidence to suggest that one or both of these shorter transcripts is the functional mRNA for °-galactosidase. All three classes of RNA were polyadenylated. Coupling of the 19S-lacZ gene to a yeast enhancer (the GAL UAS) produced a 5-fold increase in °-galactosidase activity. At the transcriptional level, activation of the enhancer resulted in a massive increase in the level of the RNA initiating at -250bp but had a minor influence of the levels of the two RNA species initiating at +9 and +25. A series of deletion mutations within the 19S promoter was constructed using Ba131 nuclease. Analysis of these mutations in yeast revealed that sequences from -500 to -193bp and from -137 to -62bp were not required for 19S promoter function, but a deletion from -62 to -21bp (which removes the putative TATA box) severely reduced 19S-1acZ gene expression. Transgenic tobacco plants containing the 19S promoter deletions fused to a CAT gene were produced by A.tumefaciens-mediated gene transfer but the analysis of these plants was not completed.