CHROMATIN MAPPING BY FLUORESCENCE IN SITU HYBRIDIZATION

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The long-term goals of this project are 1) to develop fluorescence in situ hybridization (FISH) as a tool for mapping and ordering the genome; 2) to understand the organization of sequences spaced 50 kbp to > 3 Mbp apart in chromatin during the cell cycle; and 3) to construct a long range map of Xq27-Xqter. The five specific aims of the proposed research are as follows: 1) To optimize one- and two-color detection of single copy sequences by FISH in terms of labeling efficiency, signal resolution, and image registration. 2) To compare the proximity of hybridization sites in chromatin of varying compaction. These targets include G1 interphase, prematurely condensed chromosomes, and (pro)metaphase chromosomes. The targets will be treated to extract proteins and decondense the chromatin. Emphasis will be on the selection of target(s) in which the relative spacing and order of sequences 50 kbp to 3 Mb apart can be most efficiently and accurately determined. 3) To describe the relationship between known linear distance and measured interphase and prometaphase distances for sequences separated by 50 kbp to 3 Mbp. This study will demonstrate whether FISH mapping is generally applicable to diverse chromosomal regions (telomeric vs. interstitial; G- dark vs. G-light banded). 4) To determine if the separation between sister chromatids at specific loci as cells progress from G1 to G2 can be used to derive additional map information. Sister chromatid separation will be studied in terms of the relative proximity of sequences to the telomere (human 4p16-pter) and to replication origins (DHFR region, Chinese hamster). 5) To use the FISH mapping techniques developed in aims 1-4 to construct a long range map of human chromosome Xq27-Xqter. FISH will be used a) to rapidly screen for Xq28 region-specific sequences from large-insert libraries; b) to confirm the relative spacing, orientation, and order of previously characterized PFG fragment clusters; c) to produce a fine map over regions of 1-2 Mbp by determining the spacing and order of region- specific sequences using FISH to interphase nuclei; and d) to determine if interphase distance measurements can be used to map disease loci by detecting disease-specific deletions.