The Purimidine track binding protein (or PTB) has been characterized as a RNA binding protein which appears to be involved in an alternative site selection in alternative splicing. The protein has been found to shuttle between the nucleus and the cytoplasm. We have also found that this protein preferentially forms a structure around nucleoli (which can been seen bright intranuclear dots by immunofluorescence) in HeLa cells but not in premary human fibrobrast. We believe that the dots around the nucleolus may be related to the transformation phenotype. Several other components are also found in the same structure. We interested in understanding the morphological characteristics of this structure at higher resolution using electon microscopy. Previously, using fluorescence photooxidation we have seen a plate like structure between the staining and the nucleolus. Control cells do not show the staining at the nucleolar periphery. Additional high resolution images are needed to confirm this finding. Another project that is proposed is the localization of individual RNA species in the cell nucleus by in situ hybridization. The purpose of this project is two-fold: First, we are interested in understanding the subcellular characterization of nascent RNA transcripts at their sites of synthesis. Second, we would like to determine the path that these RNAs take from the sites of trancription to the nuclear envelope, using 3-dimensional in situ hybridization and IVEM. It could either be a specific path or random diffusion through the nucleoplasm. However, there is a serious technical limitation which we have encountered previously. Due to the weakness of RNA hybridization with individual species, the eosin conjugated secondary proved too weak to be detected easily by confocal microscopy prior to photooxidation. Several improvements to the tecnique that may substantially increase sensitivity are currently being explored.
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