The cell nucleus is the repository for the genetic information of all eucaryotic cells including that 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 regulated within the confines of the cell nucleus is extremely limited. Similarly, although the genetic code behind the DNA (the nucleotide sequence) has long been broken, our understanding of the organization of this DNA into chromatin and higher order structures in the cell nucleus is still in its infancy. Progress in several areas, however, gives one reason to be optimistic about future success. Development of appropriate methods to examine the three dimensional basis of nuclear architecture coupled with new and sensitive detection systems for function, such as fluorescence in situ hybridization (FISH) and fluorescence microscopic detection of DNA replication sites are among the hopes of the future. Our laboratory has been studying the nuclear matrix and its role in DNA replication for over 15 years. Now that basic groundwork implicating the nuclear matrix as a site for the organization of DNA replication has been set, we are now focusing our attention on the 3-D organization of replication in the cell nucleus. By combining these 3-D approaches with the powerful techniques of multi-dimensional computer image analysis and FISH, we are making progress in mapping the DNA replication sites in 3-D. Use of a chromosome 11 set of DNA probes will further enable us to determine the DNA sequence organization of chromosome 11 in 3-D and map the sequences present at individual replication sites. The major directions to be pursued in this project include : (1) Study of the 3-D organization of DNA replication sites in the cell nucleus; (2) Localization of proteins at replication sites in 3-D; (3) 3-D mapping of gene sequences in the cell nucleus for human chromosome 11; (4) Map the replication of genes for human chromosome 11 in 3-D.
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