Heterochromatin is a ubiquitous yet poorly understood component of higher eukaryotic genomes. Important functional components are contained within heterochromatic regions, including ribosomal RNA genes, centromeres and telomeres. In addition, chromosome rearrangements involving heterochromatic regions are frequently found in cancerous cells. Major gaps exist in our knowledge of the nature and overall organization of DNA sequences present in heterochromatin. The molecular and genetic analyses of heterochromatin have been inhibited by the presence of large (up to megabase-size) arrays of highly repeated DNAs, or satellites. Without a greater understanding of the molecular structure of heterochromatin, our knowledge of the structure and function of higher eukaryotic genomes will remain limited. It is essential that a tractable model system is developed in a higher eukaryote that utilizes molecular and genetical approaches to circumvent the unique problems posed by the size and sequence composition of heterochromatin. Without a careful pilot study on a specific, defined heterochromatic region, it is impossible to know if current molecular genetic approaches can be successfully applied to solving the structure of heterochromatin. This research proposal focuses on studying the molecular organization of a specific heterochromatic region that constitutes 75% of a Drosophila minichromosome, Dp(1;f)1187. The presence of cloned, single-copy sequences adjacent to the Dp(1;f)1187 heterochromatin provides a direct entry point that facilitates the analysis of the highly repeated DNA. The experiments described in this proposal are designed to generate detailed structural information about the heterochromatin of Dp(1;f)1187, and to test the efficacy of new experimental approaches to the problems encountered during molecular analyses of regions rich in repeated DNA.
The specific aims of this proposal are to provide a comprehensive structural analysis of Dp(1;f)1187 heterochromatin by 1) producing a detailed restriction map with pulsed field Southern analyses; 2) generating new entry points into the proximal heterochromatin via irradiation-induced rearrangements and single P-element mutagenesis, and 3) cloning specific regions of Dp(1;f)1187 heterochromatin in high molecular weight DNA vectors, determining the stability of repeated DNA in such clones, and performing a DNA sequence analysis. The information and tools generated by studies in this model system will further our understanding of higher eukaryotic genome structure, and provide a groundwork for analysis of heterochromatin structure and function in other eukaryotes, including humans.
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