|M.Sc Student||Cohen Yotam|
|Subject||Identifying Locations of RNA polymerase II along the|
Genome in Stationary-phase Cultures of the Yeast
S. cereuisiae, under Various Stress
Conditions, Using a ChIp-on-
|Department||Department of Biotechnology and Food Engineering||Supervisor||Professor Yechezkel Kashi|
|Full Thesis text|
The association between transcription and DNA repair is acknowledged as a player in the generation of mutations in a non-random fashion in prokaryotes and eukaryotes. The transcription complex is capable of directing DNA repair to sites of transcription, which may lead to mutations preferentially in transcribed genes.
Using microarray analysis of growth-arrested yeast cultures, Aviv de Morgan and co-workers demonstrated the co-localization of a DNA-turnover marker, indicative of DNA-repair-associated DNA synthesis, with genes persistently transcribed during stationary phase. For precise picture, measuring mRNA levels in the cell is not enough, since differences in mRNA stability influence mRNA level in the cell. In addition, stationary phase yeast have large P-bodies containing mRNAs that re-enter translation when growth resumes.
Our goal was to find the locations of RNAPII along the genome in response to osmotic stress in stationary state cultures in order to verify the transcription pattern in response to this specific stress. We grew cultures to mid-log phase, and then transferred them to minimal medium without any carbon source with and without 0.25M NaCl. From that time point we started to sample the cultures.
The samples were fixed using formaldehyde, cells were broken using bead-beater and the chromatin was sheared using sonication. DNA fragments attached to RNAPII in his two forms (phosphorylated and non-phosphorylated) were specifically immunoprecipitated. The immunoprecipitated DNA was amplified and labeled using PCR and hybridized to microarrays. The hybridization results were statistically analyzed for testing the salt effect on the RNAPII locations.
The results we have got from the hybridization analysis, though not unambiguous, indicated a cell response to the osmotic stress. Many genes connected to osmotic stress were up regulated and the RNAPII binding to these genes increased. Other genes, connected to DNA repair processes such as transcription coupled repair (TCR) genes were up-regulated too. In contrast, genes that are not important to the cell in stress condition, such as replication genes, and etc., showed decrease in the RNAPII binding levels. Nonetheless, five genes known as stress related which we expected to show an increase in the RNAPII binding level, showed opposite behavior and were down regulated. A further work should be done to verify this result.
In order to complete this research, one should repeat these experiments, looking for the RNAPII locations, DNA repair pattern and transcription levels on the same culture using different stress conditions.