Bloom syndrome(BS) is an autosomal recessive, spontaneous chromosomal instability disorder demonstrating a clinical constellation which includes severe proportional dwarfing; sun-sensitive telangiectatic erythema; and a significant predisposition to the development of leukemia, with early onset of other neoplasias. Cytologically, the pathognomonic phenotype is a many- fold increase in the spontaneous rate of sister chromatid exchanges (SCE), elevated frequencies of chromosomal breakage and the appearance of quadriradial figures. Although BS cells exhibit normal responses for most DNA repair assays which have been studied, deficiencies and/or abnormalities have been found in the activities of both DNA ligase I and uracil DNA glycosylase. Taken collectively, enzymatic data would support a processing or regulation defect, rather than a primary mutation in either DNA ligase I or uracil DNA glycosylase. Whole-cell fusion experiments have demonstrated complementation of the elevated SCE rates in BS cells to normal frequencies. However, such investigative approaches are limited by lack of selective methods to specifically identify and stabilize hybrid cells resulting from those fusions, compromising efforts to accurately evaluate complementation. The present application proposes an alternative genetic approach to quantitatively characterize the correction of high SCE phenotype in BS fibroblasts and to chromosomally localize the complementing gene, leading to its molecular cloning. Individual normal human chromosomes will be transferred to BS cells by microcell mediated chromosome transfer (MMCT). The MMCT process will be facilitated by the use of an established collection of mouse/human hybrids bearing human chromosomes """"""""tagged"""""""" with a dominant selectable marker (neo). Following MMCT and selection, each clone arising will be independently screened for correction of high SCE levels and the chromosome responsible for complementation will be identified with cytogenetic and molecular techniques. Complemented clones will be quantitatively characterized for BS associated cellular phenotypes, including rates of chromosome breakage, and the appearance of both DNA ligase I activity and the normal antigenic form of uracil DNA glycosylase. Cytogenetically detectable deletions and rearrangements of the complementing chromosome will be used to achieve finer mapping of the complementing locus. Quantitation of phenotypic complementation, fine mapping of the locus rendering correction, and the establishment of rodent/human hybrids bearing complementing and noncomplementing deviations of that single human chromosome, will provide the materials necessary for molecular cloning and characterization of the gene involved in BS complementation. The proposed study will provide valuable information leading to the ultimate identification of the specific protein defect associated with elevated SCE rates in BS cells and a better understanding of events which predispose to neoplasia in this syndrome.
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