GENE MAPPING/ISOLATION BY CHROMOSOME TRANSFER AND POSITIONAL CLONING

克隆新的FA基因有几种可能的策略。Cdna DNA交联型超敏反应的互补作用 导致了一个FA基因的克隆。然而，这种方法是有限的。 根据两个因素：a)cdna必须相对较小，b)适当 基因的调节表达不一定是细胞生存所必需的。 分离FA基因的另一种方法是定位克隆。到期 事实上，存在着多个互补小组，而且 家系的数量相对有限，FA基因的定位通过 单靠遗传连锁研究似乎很困难 我们建议使用染色体转移来定位FA的位置 总水平的基因，其次是遗传连锁和物理作图 进一步细化染色体位置，并最终克隆 基因。 标记了0418抗性基因的人类染色体将被 转移到FA患者转化的成纤维细胞中， 已知不代表互补C组。用DNA进行选择 交联剂将识别互补克隆和染色体 赋予抵抗力量的身份将被确定。更正的大小 在这种方法中，cDNAs是不相关的，并且适当地调节基因在 文中还提出了问题。一旦一条互补的染色体 确定后，FA基因的定位就越准确 体细胞杂交种的缺失作图鉴定及遗传 联动。现有的标记以及由染色体产生的新标记 将使用显微解剖和聚合酶链式反应进行遗传图谱的绘制。这一步 之后是将YAC与关键区域物理隔离 表达序列的作图和克隆。候选的cDNA将是 通过化学错配裂解筛选可能的突变。 要使用的另一种方法是识别鼠标 染色体携带如上所述的FA基因。补充FA细胞 小鼠的DNA可用于制备消减文库和 FA候选基因的直接克隆。

Several possible strategies for the cloning of new FA genes exist. cDNA complementation of the DNA cross-linking hypersensitivity has already resulted in the cloning of one FA gene. This method, however, is limited by 2 factors: a) the cDNA has to be relatively small and b) properly regulated expression of the gene must not he essential for cell survival. An alternative method for isolating FA genes is positional cloning. Due to the fact, that multiple complementation groups exist and that the number of families is relatively limited, the mapping of the FA genes by genetic linkage studies alone appears difficult We here propose to use chromosome transfer to map the location of FA genes at a gross level, followed by genetic linkage and physical mapping to further refine the chromosomal position and to ultimately clone the genes. Human chromosomes marked with the 0418 resistance gene will be transferred into transformed fibroblasts from FA patients, which are known to not represent complementation group C. Selection with DNA crosslinking agents will identify complemented clones and the chromosome conferring the resistance will be identified. The size of the correcting cDNA is not relevant in this method and proper regulation of the gene in question is also given. Once a complementing chromosome has been identified, the more precise localization of the FA gene will be identified by deletion mapping in somatic cell hybrids and genetic linkage. Existing markers as well as new markers generated by chromosome microdissection and PCR will be employed for genetic mapping. This step will be followed by isolation of YACs from the critical region, physical mapping and the cloning of expressed sequences. Candidate cDNAs will be screened by chemical mismatch cleavage for possible mutations. An additional approach to be used is the identification of mouse chromosomes bearing FA genes as described above. FA cells complemented by mouse DNA could then be used to prepare subtractive cDNA libraries and direct cloning of FA candidate cDNAs.