We are investigating the feasibility of developing clonable electron-dense labels that can be visualized in three-dimensional reconstructions of subcellular organelles obtained by electron tomography. Currently, electron tomography is often limited by uncertainty in determining which proteins of interest correspond to specific features in the reconstruction. Clonable labels could potentially localize those proteins, by analogy to green fluorescent protein in optical microscopy. Bacterial ferritin, an iron storage protein that accumulates a dense iron oxide nanoparticle in its hollow core, is a promising candidate. A modified ferritin gene from Listeria innocua was cloned into Escherichia coli so that the inserted DNA coded for clusters of ferritin subunits connected by flexible linker sequences. Constructs with 2,4,8 and 12 linked ferritin subunits were synthesized and cloned. Currently we are optimizing conditions for the in vivo iron-loading of ferritin into E. coli. ? ? Another approach to expressing clonable markers involves the development of functional RNA labels. In collaboration with Dr. Bruce Shapiro (NCI, Frederick), DNA coding for Nanoring and Nanotube RNA has been cloned into E. coli. In order to express this structure, a T7-driven expression vector for a modified version of the Nanoring has been developed. To obtain the RNA required for self-assembly the Nanoring RNA is flanked by two ribozymes (Hammerhead ribozyme and Varkud satellite ribozyme). After in vivo cleavage by these two ribozymes the Nanoring monomer is generated, which can self-assemble into rings and tubes. Currently expression and assembly conditions of this system are being optimized.
