The long term goal is to develop new flow cytometric (FCM) assays that utilize multiple, DNA base-specific fluorochromes and advanced FCM technology a) to detect subtle changes in the native configuration of chromatin in response to physiological stimuli and b) to improve resolution of individual chromosomes. The rationale for these studies is based on the concept that modulations in chromatin structure are brought about by alterations in the complex interaction between DNA and nucleoproteins. These alterations produce changes in DNA accessibility to base-specific fluorochromes that can be efficiently detected by FCM. Correlated analysis of the binding by multiple fluorochromes having different modes for binding to DNA can provide a unique approach for assessing changes in chromatin organization. Such new technology provides distinct advantages for clinical, structural and cell biology studies including, a) the application of DNA fluorochromes to cells or chromosomes under relatively non-perturbing conditions so that chromatin remains close to the native configuration, b) rapid FCM analyses for correlating the relative changes in binding by multiple fluorochromes in cells and c) requirement of small numbers of cells. To achieve their long range goal they have listed the following specific aims: 1) to develop methods for multi-fluorochrome staining of DNA in cells and chromosomes, employing combinations of dyes that have unique spectral, lifetime and base-pair binding characteristics for sensitive and accurate labeling of particular regions on DNA, 2) to evaluate and optimize analytical techniques for use in conventional and unique FCM systems to correlate DNA-fluorochrome probe interactions with modulations in chromatin structure, and 3) to apply this new FCM technology for analysis of progressive changes in chromatin structure in selected biological systems and for characterization of individual murine and human chromosomes. They propose to take advantage of progress made during the previous grant period and expertise in our laboratory for cell-cycle analysis, chromatin structure, nucleoprotein biochemistry, DNA-damage mechanisms and chromosomes. Additionally, They will utilize FCM systems available through the National Flow Cytometry Resource at Los Alamos, including a unique flow system which measures fluorescence lifetime in conjunction with conventional FCM measurements. The applicant states the proposed studies will lead to the development of novel cytochemical approaches for correlating chromatin structure and cellular function not previously obtainable by flow cytometry or other analytical systems.
