Poster abstracts
Poster number 110 submitted by Luke Parks
Fluorescent microscopy of transcription factories by BrU and EdU labeled foci
Luke Parks (Department of Radiation Oncology, University of Michigan), Michelle Paulsen (Department of Radiation Oncology, University of Michigan), Mats Ljungman (Departments of Radiation Oncology and Environmental Health Sciences, University of Michigan)
Abstract:
Replication and transcription must often operate simultaneously, though the exact nature of this interplay is not fully delineated. Within the nucleus are thousands of RNA polymerases (RNAPol). It has been theorized that the RNAPol may aggregate to form what is referred to as transcription factories. These factories constitute hundreds of RNAPol complexes and may be fixed in location. Conversely, DNA polymerases (DNAPol) are also thought to be fixed to certain locations in the nucleus. By using high resolution fluorescent microscopy and short pulse-labeling of nascent RNA by bromouridine followed by immunocytochemistry using anti-BrdU antibodies, we visualized active transcription in individual human fibroblasts. Our results show about 200 labeled foci per cell representing active transcription factories. We are currently using EdU Click-iT to label nascent DNA in tandem with bromouridine-labeling of nascent RNA, to get a snapshot of the simultaneous nuclear localization of both DNA and RNA synthesis. We hypothesize that the coordination of transcription and replication is aberrant in cancer cells leading to replication stress and genomic instability.
Keywords: Transcription factories, Nascent RNA, Replication