| Budget Amount *help |
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1986: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1985: ¥4,900,000 (Direct Cost: ¥4,900,000)
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| Research Abstract |
We have previously established that typical Fanconi's anemia (FA) cells have a characteristic defect in the first half-excision of mitomycin C (MMC)-induced interstrand cross-links in the DNA and exhibit the supersensitivity to the lethal and clastogenic effects of MMC. To attempt to clone the human repair gene complementing the FA defect, the present study employed the transfection of HeLa genomic DNA and total cDNA to FA fibroblasts and SV40-immortalized FA cell line.
First we employed DNA (HeLa S3 genome)-mediated gene transfer to FA14JTO fibroblast cells and selected twice with MMC. Among many transformants, we obtained a single stable, well-growing clone designated FA14t113, althoug all other clones were eventually senesced. FA14t113 clone cells were as resistant to MMC killing as normal human cells and were able to perform the first half-excision of MMC cross-links in the normal fashion with a half-life of 2 h, in contrast to the defective parental FA14JTO cells. in addition, we o
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btained no MMC-resistant revertants of FA14JTO cells in the identical large-scale experiments. Thus, we succeeded in the DNA-mediated gene transfer for FA fibroblast. However, the next trial that invloves the transfection of neo-gene-ligated Sal I fragments (about 40 kb) of HeLa DNA provided no clones of MMC-resistance and neo-resistance.
Second, we transfected, therefore, the total DNA of newly constructed cDNA library (pcDHF) to SV40-immortalized FA GM6914 cells. From large-scale experiments, many partially MMC-resistant clones were obtained after two selections with MMC. Further screening to eliminate unstable clones gave the 4 final stable clones, among which an FApcD5102-8 clone had the most MMC-resistant phenotype, despite a partial resistance ( 5 times more than parental GM69148 but 3 time less than normal), and a single pcD integration in the Southern blot. To clone such a cDNA-containing fragment, 20 kb Sau3AI fragments of FApcD5102-8 DNA were ligated to the EMBL4 cloning vector, and after in vitro packaging, we selected 8 positive phage clones by plaque hybridization. Each phage DNA was amplified and transfected to GM6914 cells to select the desired clone. Finally we obtained a single EMBL phage clone that conferred MMC resistance to GM6914 cells similar to that of FApcD 5102-8 clone. In conclusion, we have succeeded in the molecular cloning of a human cDNA gene that confers the MMC resistance to FA cells. In future, we can determine the base sequence to characterize the gene product and search for genomic gene and its localization. Less
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