CAFFEINE-ENHANCED ANALYSIS OF CHROMOSOME BAND 11Q23

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This project has two long-term objectives: 1) to develop an improved simple high resolution banding (HRB) technique for the detailed cytogenetic analysis of human chromosomes; and 2) to precisely define breakage points in the 11q23 chromosomal region in a library of widely-available human cell lines for use by other investigators. A simple and efficient HRB technique would be medically valuable both in clinical diagnosis of inherited chromosomal disorders and spontaneous tumors, and in medical research in human gene mapping and chromosomal mutation. The 11q23 region is medically significant for its breakage in some myeloid leukemias, and the locus of the ets-1 proto-oncogene and a fragile site, and as the probable locus for several other genes. A library of cell lines with precisely-localized 11q23 breakpoints would be useful to investigators for rapid gene mapping and molecular analysis of this important chromosomal region. This grant proposal presents a two-phase experimental plan to achieve these objectives. The first phase will test the hypothesis that caffeine increases the efficiency of high resolution banding techniques utilizing actinomycin-D, acridine orange, 5'- bromodeoxyuridine or ethidium bromide, in the trypsin G-banding analysis of four human fibroblast and lymphoid cell lines. If caffeine is shown to increase the mitotic index and the proportion of cells with high resolution banded chromosomes, then a simple caffeine-enhanced protocol will be designed for use on cultured human cells. The reversibility of these caffeine effects by cycloheximide will also be tested. The second phase will apply this improved HRB technique to the detailed analysis of 11q23 translocations already in 9 human cell lines in the NIGMS Human Genetic Mutant Cell Repository. High resolution banding analysis will enable identification of the specific sub-band involved in each line's 11q23 translocation, as well as detailed karyotyping of each cell line.