ISOLATION OF THE DEB-SENSITIVITY GENE OF FANCONI ANEMIA

范可尼贫血（FA）FA是一种常染色体隐性遗传疾病， 儿童期的特点是多方面的发展， 影响骨骼和肾脏系统的异常， 恒定和致死性全血细胞减少症，以及增加的 易患肿瘤性疾病，尤其是骨髓性白血病。 来自FA患者的培养细胞表现出自发性增加， 与正常细胞相比，染色体断裂。 他们也是 对DNA的细胞杀伤和致染色体断裂作用过敏 交联剂和烷基化剂如丝裂霉素C（MMC） 和二环氧丁酸酯（DEB）。 我们已经发展出了一种克隆生存 一种能够明确区分正常 和基于DEB超敏反应的FA细胞。 以此作为 选择性系统，我们已经证明，无论是细胞和 FA细胞对DEB处理的染色体超敏性可 通过转染人胎盘或 磷酸钙沉淀的中国仓鼠DNA。 因此 补充FA的关键细胞表型的基因 存在于人类和中国仓鼠的DNA中。 此外，它们在转染后稳定整合和表达 转化成变异细胞 如果修复缺陷被证明是主要缺陷 这种疾病的细胞病变，然后克隆和表征 不仅对理解基因的功能， 这种疾病的分子基础， 最终在体内纠正造血干细胞的策略 缺损 该项目的总体目标是克隆和表征 导致DNA交联的FA缺陷基因， 烷化剂超敏反应，具体如下 目的：（i）分离涉及抗性的连锁转染 人类和仓鼠的基因来源和共同的新基因， gpt基因使用后者作为表型和基因型标记， 第二轮和后续的转染。 (ii)隔离双 来自抗性细胞系的微小染色体（DM）DNA， 利用凝胶内复性技术表达DM， 将扩增的序列放回突变细胞中， 这些确实代表了扩增的抗性基因。 (iii)隔离 仓鼠来源的抗性决定序列的筛选 来源于仓鼠DNA的DEB菌株的基因组文库 用标记的仓鼠DNA作为探针转染。 (iv)隔离 利用cDNA的消减杂交， 从经受慢性DEB的正常细胞构建的文库 暴露和过量的poly A+ mRNA从未处理的正常和 FA细胞。 (v)进行详细的分子表征 抗性基因的结构和功能。

Fanconi anemia (FA) FA is an autosomal recessive disorder of childhood which is characterized by multiple developmental anomalies that affect the skeletal and renal systems, an invariable and lethal pancytopenia, and an increased predisposition to neoplastic disease, especially myeloid leukemia. Cultured cells from FA patients exhibit increased spontaneous chromosome breakage compared to normal cells. They also are hypersensitive to the cell killing and clastogenic effect of DNA cross-linking and alkylating agents such as mitomycin C (MMC) and diepoxybuate (DEB). We have developed a clonogenic survival system that allows unambiguous discrimination between normal and FA cells based on DEB hypersensitivity. Using this as the selective system, we have demonstrated that both the cellular and chromosomal hypersensitivity of FA cells to DEB treatment can be completely corrected by transfection of human placental or Chinese hamster DNA as calcium phosphate precipitates. Thus, a gene or genes that complement the key cellular phenotypes of FA are present in both human and Chinese hamster DNA. Furthermore, they stably integrate and express upon transfection into mutant cells. If the repair defect turns out to be the primary cellular lesion of this disorder, then cloning and characterization of the responsible gene(s) is significant not only for understanding the molecular basis of the disorder, but also for planning strategies for eventual in vivo correction of the hematopoietic defect. The overall aim of this project is to clone and characterize the gene(s) defective in FA that lead to DNA cross-linking and alkylating agent hypersensitivity with the following Specific Aims: (i) Isolate linked transfections involving the resistance genes of human and hamster derivation and the co- transfect neo- gpt genes using the latter as phenotypic and genotypic markers in second and subsequent rounds of transfection. (ii) Isolate double minute chromosome (DM) DNA from resistant cell lines that express DMs using the in-gel renaturation technique and transfect the amplified sequences back into mutant cells to determine if these indeed represent amplified resistance genes. (iii) Isolate resistance determining sequences of hamster origin by screening the genomic library of a DEB strain derived from hamster DNA transfection with labelled hamster DNA as the probe. (iv) Isolate resistance genes by using subtractive hybridization of a cDNA library constructed from normal cells subjected to chronic DEB exposure and excess poly A+ mRNA from untreated normal and FA cells. (v) Undertake detailed molecular characterization of the structure and function of the resistance gene(s).