It had been suggested that transient torsional stress may be required for transcription of chromatin templates in vivo. To investigate this issue, we have developed an in vivo experimental system in yeast to study whether template linearization either upstream or downstream of a transcription unit, CUPl-lacZ. could inhibit transcription from the reporter lacZ gene. The technique uses yeast HO endonuclease, a sequence-specific double-stranded DNA nuclease, for targeting DNA cleavage. Experiments were performed with cells carrying two yeast CEN-based episomal plasmids, one containing an endonuclease gene under the control of the GAL 10 promoter and the other bearing a reporter fusion gene with a HO cut site at preselected positions. We first arrested yeast harboring these plasmids at the G l phase and then induced HO endonuclease synthesis by adding galactose. When template DNA was efficiently linearized (about 80% of total), copper was added to the culture to induce lacZ expression. The levels of lacZ transcript were then measured by a RNase protection assay. Our results revealed that transcription from linearized templates was as efficient as that from templates containing intact DNA, with essentially identical kinetics of induction. Similar results were obtained for the same reporter genes integrated into the LYS2 locus on yeast chromosome II. Therefore, in direct contrast to expectations from earlier reports, in vivo linearization of DNA templates does not have any negative effects on transcription in yeast.
|Original language||English (US)|
|State||Published - 1996|
ASJC Scopus subject areas
- Molecular Biology