| The aim of this thesis was to gain a better understanding of the transcriptional regulation and posttranslational modification of the eukaryotic translocation factor, elongation factor 2 (EF-2) in Saccharomyces cerevisiae. Toward this end, I examined the biosynthesis of the unique amino acid diphthamide that is the target for diphtheria toxin (DT) and is found only in EF-2, and I have analyzed the transcriptional regulation of the two yeast genes for EF-2.;Two different selection schemes, a genetic and a biochemical scheme, were developed for cloning the diphthamide biosynthetic genes. Both selection schemes were based on complementation of yeast mutants that were resistant to diphtheria toxin because they lack a functional enzyme of the diphthamide biosynthetic path. In both cases the candidates are chosen based on their ability to render the mutants sensitive to DT. The source of DT in the genetic selection scheme was an intracellular plasmid that expresses the DT-A fragment. This selection produced a high number of false positives which, in combination with other limitations, led me to develop a second, biochemical, selection scheme. The candidates from the biochemical scheme are assayed by the in vitro, DT-mediated, ADP-ribosylation reaction. Using the biochemical scheme, we have generated positive preliminary results which indicate that this selection will produce the desired genes.;The second part of my thesis involves mapping the promoters of the two genes for EF-2 in yeast, EFT1 and EFT2. Each gene has a single, major transcription start site and several minor start sites. Both promoters contain poly(T) rich regions and potential UAS and TATA sequences. Using lacZ fusions to the promoters of each gene as a reporter system, I have determined that EFT1 and EFT2 are differentially expressed and I have mapped the upstream activating sequences of both genes. The EFT2 promoter-lacZ fusion gives a 2.5-3.0 fold higher level of b-galactosidase activity than does the EFT1 promoter-lacZ fusion. Examination of the mRNA levels of the two genes confirm the b-galactosidase results and show that there is a 2.0-2.5 fold higher level of EFT2 mRNA than EFT1 mRNA in a cell containing a functional copy of both genes. |
