NUCLEAR MATRIX STRUCTURES AND GENOMIC FUNCTION

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The cell nucleus is the repository for the genetic information of all eucaryotic cells including those of man. Despite considerable progress in defining basic molecular properties of the primary genomic functions of DNA replication, transcription, and RNA splicing and processing, our knowledge of how these processes are organized and coordinated within the confines of the functional cell nucleus has been until recently very limited. Impressive progress in applying molecular probes along with developments in 3-D microscopy and computer image analysis is rapidly correcting this deficiently and is leading to a new view of the cell nucleus based on the functional dynamics of nuclear architecture. In situ labeling coupled with three dimensional microscopy and computer imaging techniques reveal that DNA replication and transcription sites are organized into higher order units or "zones" in the cell nucleus. The dynamic interplay and "re-zoning" of replication and transcription regions during the cell cycle may form the structural basis for the elaborate global coordination of replicational and transcriptional programs in the mammalian cell. As one basis for this functional organization, the genome is arranged in discrete chromosome territories in the interphase nucleus. This organization is strikingly maintained following extraction of cells for nuclear matrix under conditions which maintained the DNA intact. Appropriate extraction of the nuclear matrix internal components leads to a corresponding disruption of chromosome territories along with the release of a discrete subset of proteins that are candidates for being chromosome territory anchoring proteins. With these previous studies as a basis we propose the following specific aims: (1) Further develop multi-dimensional computer imaging approaches to 3-D microscopy; (2) Elucidate the 3-D spatio-temporal properties of DNA replication and transcription sites for genes replicating in early S-phase; (3) Study chromosome territories in relations to higher order domains of DNA replication and transcription sites and characterize candidate chromosome territory anchor proteins; (4) Investigate the 3-D dynamics of DNA replication and transcription sites in living cells.