This is a revised application of a competitive renewal requesting three years of support to study two genes, recently isolated in the principal investigator's laboratory, that when defective lead to Fanconi anemia (FA). FA is an autosomal recessive disorder characterized by severe bone marrow failure, congenital malformations, and susceptibility to the development of certain malignancies, in particular, acute myelogenous leukemia. FA cells are hypersensitive to certain cytotoxic and clastogenic agents, for example, mitomycin C (MMC) and diepoxybutane (DEB) and FA cells exhibit chromosome aberrations. The long-term objectives of this research is to elucidate the biological basis of FA in order to gain insights into chromosome stability, cell growth, development and cancer. There are five complementation groups in FA defined by cell hybridization studies (FA-A through E). Previously, only one FA gene had been isolated, FAC. The encoded product is not homologous to any other known amino acid sequence and, to everyone's surprise, is localized to the cytoplasm. The applicant shows now that, using the same expression cloning procedure he used to isolate FAC, he has isolated the FAA gene and confirmed its identity by the identification of mutations in FA-A cells; he also has a strong candidate for the FAD gene. He also presents good evidence that the FA-B complementation group is a subset of the FA-A group, because the FAA cDNA complements the MMC and DEB sensitivity of FA-B cells. The applicant proposes that intragenic complementation results when FA-A and FA-B cell lines are fused. Like FAC, the two novel gene products of FAA and FAD have no homologies to known amino acid sequences. The applicant now proposes to characterize the FAA and FAD genes and their gene products.
The first aim i s to confirm the identity of the FAD cDNA by determining the presence of mutations in FA-D cells.
His second aim i s to test the hypothesis that FA-B is a subset of FA-A, by determining the presence of mutations in FA-B cells (two cell lines are known). Intragenic complementation will be investigated by introducing FAB and FAA mutations into cDNA expression constructs, co-transfecting the FAB and FAA mutants into FA-A cells and FA-B cells, respectively, and then testing the transfectants for MMC and DEB sensitivity.
The third aim i s to characterize the FAD gene organization, analyze the RNA products of FAD by RACE and primer extension, and examine the distribution of expression by multiple-tissue Northern blot and analysis by in situ hybridization of sections of mouse tissue using the cloned mouse gene. The fourth and most ambitious aim is to investigate the functional relationship of the FAA, FAD and FAC gene products. He will (1) raise antibodies to FAA and FAD products to conduct indirect immunofluorescence studies; (2) identify interactions between FA proteins (homotypic and/or heterotypic) by immunoprecipitation studies of metabolically labeled cells, by the yeast two-hybrid approach, by Far-Western detection, and by FA-protein affinity chromatography; (3) identify the function of the protein by (a), if nuclearly localized, testing potential enzymatic activities related to DNA metabolism (e.g., polymerase, nuclease, helicase, etc.) or (b), if cytoplasmically localized, asking whether FA protein functions in apoptosis by overexpressing FAA and FAD in two growth-factor dependent cell lines and testing whether cell death is delayed upon removal of growth factor. The fifth aim is to isolate the FAE gene by the expression cloning used so successfully to date.
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