Eccleston, Patricia Alexandra; (1993) Transcription factors regulated by the adenovirus E1a gene. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In this thesis, the yeast S. cerevisiae has been used to characterise the interaction of transcription factors with other proteins and with DNA. This involved three studies. Firstly, the mechanism of transcriptional activation by the adenovirus E1a gene was investigated. The initial goal was to reproduce E1a activation in yeast and subsequently take advantage of yeast genetics to analyse the mechanism in detail. The transcriptional process in yeast and mammalian cells is highly conserved. Despite this, I showed that expression of E1a in yeast was unable to modulate transcription mediated by yeast factors. One mammalian transcription factor shown to mediate Ela transactivation is ATF2. It had been proposed that a direct physical interaction occurs between the two proteins, so I investigated this possibility using a yeast genetic system. No such interaction was seen, although as a control, E1a was seen to interact with the retinoblastoma gene product. An ATF binding activity is present in yeast itself. The second study used a yeast screen with an activation domain tagged cDNA library in order to isolate the yeast ATF transcription factor. Clones were isolated that specifically activated an ATF site. However, they may be involved in the regulation of this site, rather than encode the ATF transcription factor itself. E2F is another mammalian transcription factor that has been shown to mediate E1a transactivation. The interaction of E2F with a number of cellular proteins may modulate its activity in a cell cycle dependent manner. One result of adenovirus infection is the dissociation of these complexes by E1a and the interaction of E2F with a viral E4 gene product. The third study involved an attempt to isolate an E2F cDNA. A yeast genetic screen was set up to isolate cDNA clones that could either bind directly to an E2F DNA binding site or bind to the adenovirus E4 protein. Screens for direct binding to the E2F site did not yield any positive clones, possibly because such a screen would depend upon E2F binding as a homodimer. A screen using the E4 protein yielded clones whose products could interact with E4 in yeast. Preliminary analysis of these clones has been carried out in in vitro systems and in mammalian cells.

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