This proposal addresses the question of how chromatin is organized in the eukaryotic nucleus, by examining a novel hypothesis. The hypothesis is that proteins associated with the telomeres, or ends, of chromosomes (telomeric proteins, or TP) are nuclear lamins, which in turn are members of a large group of proteins called intermediate filaments (IF). Their ability to assemble and disassemble into filaments would then be responsible for maintenance of nuclear organization. The organism to be used in these studies is the hypotrichous ciliate Euplotes eurystomus, which is particularly advantageous because of the unusually short lengths of individual chromosomal elements in the macronuclei, resulting in approximately 100,000,000 telomere-bearing DNA pieces, in which the telomeric DNA sequences are all the same. Furthermore, the macronucleus contains clearly defined regions of condensed, dispersed, and replicating chromatin. The higher order organization which determines the structure of each of these functional regions is not known. These properties make Euplotes an excellent system for the study of telomeres and eukaryotic nuclear structure and function. The experiments proposed for this study involve characterization of TPs by cloning the gene and determining its sequence. The sequence will be compared with sequences of known IFs. Immunofluorescent localization and STEM mass determination will be used to determine TP nuclear sites and stoichiometry with DNA. In situ electron microscopic analysis and immunolocalization will be used to characterize the 10 nm filaments. Reconstitution experiments will be performed to determine factors which affect chromatin organization. It is expected that this study will clarify one of the functions of telomeres and the role of lamins in organizing chromatin structure in a eukaryotic nucleus. Thus, the project addresses one of the central issues of cell biology, namely, the architectural organization of the genome within the nucleus.
