The long term goal of this work is a better understanding of the components that mediate, and regulate, transcription in eukaryotic cells. Transcriptional regulation plays an important role during such essential processes as differentiation and development. Detailed knowledge of the repetoire of mechanisms whereby transcription can be regulated provides an essential background to elucidation of the molecular events that govern such normal processes, as well as to the abnormal behaviour displayed by oncogenic cells. In the proposed studies, two transcriptional systems will be examined in vitro. A fraction obtained from HeLa cells that is required for accurate transcription from a template that contains no TATAsequence element has been identified using the adenovirus type 2 (Ad2) major late and IVa2 control regions that do and do not, respectively, contain a TATA element. This fraction will be further purified to determine whether it contains one, or more, factor(s) and to permit the biochemical properties of the factor(s) to be investigated. The ability of the factor(s) to induce accurate transcription from additional promoter sites that do not contain TATA elements will be tested in reconstituted in vitro transcription systems. Specific DNAbinding activity of the factor(s) will be assessed using several assays and the mechanism whereby the factor(s) promote specific transcription will be investigated. Adenovirus type 2 infection induces enhanced transcriptional activity in whole cell extracts. The transcriptional activities of extracts of HeLa cell will be compared to those displayed by extracts made from cells expressing various combinations of the viral E1A and E1B proteins to confirm the results of preliminary experiments that suggest that enhanced transcriptional activity in vitro reflects the activity of the E1A 289R proteins, known to induce stimulated transcription of viral and certain cellular genes in vivo. Extracts prepared from Ad2infected cells will be fractionated on various chromatographic matrices and the fractions assayed for stimulatory activity. The molecular mechanism of stimulation of transcription will be investigated, as will the mechanism whereby the E1A 289R proteins induce enhanced transcriptional activity.
Showing the most recent 10 out of 13 publications
